Compartmentalised combinatorial chemistry by microfluidic control

ABSTRACT

The invention describes a method for the synthesis of compounds comprising the steps of: (a) compartmentalising two or more sets of primary compounds into microcapsules; such that a proportion of the microcapsules contains two or more compounds; and (b) forming secondary compounds in the microcapsules by chemical reactions between primary compounds from different sets; wherein one or both of steps (a) and (b) is performed under microfluidic control; preferably electronic microfluidic control The invention further allows for the identification of compounds which bind to a target component of a biochemical system or modulate the activity of the target, and which is co-compartmentalised into the microcapsules.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/962,952, filed Oct. 12, 2004, which claims priority under 35 U.S.C. §120, to PCT Application No. GB2004/001352 filed Mar. 31, 2004, theentirety of which is incorporated herein by reference.

The present invention relates to methods for use in the synthesis andidentification of molecules which bind to a target component of abiochemical system or modulate the activity of a target.

Over the past decade, high-throughput screening (HTS) of compoundlibraries has become a cornerstone technology of pharmaceuticalresearch. Investment into HTS is substantial. A current estimate is thatbiological screening and preclinical pharmacological testing aloneaccount for 14% of the total research and development (R&D) expendituresof the pharmaceutical industry (Handen, Summer 2002). HTS has seensignificant improvements in recent years, driven by a need to reduceoperating costs and increase the number of compounds and targets thatcan be screened. Conventional 96-well plates have now largely beenreplaced by 384-well, 1536-well and even 3456-well formats. This,combined with commercially available plate-handling robotics allows thescreening of 100,000 assays per day, or more, and significantly cutscosts per assay due to the miniaturisation of the assays.

HTS is complemented by several other developments. Combinatorialchemistry is a potent technology for creating large numbers ofstructurally related compounds for HTS. Currently, combinatorialsynthesis mostly involves spatially resolved parallel synthesis. Thenumber of compounds that can be synthesised is limited to hundreds orthousands but the compounds can be synthesised on a scale of milligramsor tens of milligrams, enabling full characterisation and evenpurification. Larger libraries can be synthesised using split synthesison beads to generate one-bead-one compound libraries. This method ismuch less widely adopted due to a series of limitations including: theneed for solid phase synthesis; difficulties characterising the finalproducts (due to the shear numbers and small scale); the small amountsof compound on a bead being only sufficient for one or a few assays; thedifficulty in identifying the structure of a hit compound, which oftenrelies on tagging or encoding methods and complicates both synthesis andanalysis. Despite this split synthesis and single bead analysis stillhas promise. Recently there have been significant developments inminiaturised screening and single bead analysis. For example, printingtechniques allow protein-binding assays to be performed on a slidecontaining 10,800 compound spots, each of 1 nl volume (Hergenrother etal., 2000). Combichem has so far, however, generated only a limitednumber of lead compounds. As of April 2000, only 10 compounds with acombinatorial chemistry history had entered clinical development and allbut three of these are (oligo)nucleotides or peptides (Adang andHermkens, 2001). Indeed, despite enormous investments in both HTS andcombinatorial chemistry during the past decade the number of new drugsintroduced per year has remained constant at best.

Dynamic combinatorial chemistry (DCC) can also be used to create dynamiccombinatorial libraries (DCLs) from a set of reversibly interchangingcomponents, however the sizes of libraries created and screened to dateare still fairly limited (≦40,000) (Ramstrom and Lehn, 2002).

Virtual screening (VS) (Lyne, 2002), in which large compound bases aresearched using computational approaches to identify a subset ofcandidate molecules for testing may also be very useful when integratedwith HTS. However, there are to date few studies that directly comparethe performance of VS and HTS, and further validation is required.

Microfluidic technology has been applied to high throughput screeningmethods. For example, U.S. Pat. No. 6,508,988 describes combinatorialsynthesis systems which rely on microfluidic flow to control the flow ofreagents in a multichannel system. U.S. Pat. No. 5,942,056, andcontinuations thereof, describes a microfluidic test system forperforming high throughput screening assays, wherein test compounds canbe flowed though a plurality of channels to perform multiple reactionscontemporaneously.

Despite all these developments, current screening throughput is stillfar from adequate. Recent estimates of the number of individual genes inthe human genome (˜30,000) and the number of unique chemical structurestheoretically attainable using existing chemistries suggests that anenormous number of assays would be required to completely map thestructure-activity space for all potential therapeutic targets (Burbaum,1998).

Hence, a method with the capability to both create and screen vastnumbers (≧10¹⁰) of compounds quickly, using reaction volumes of only afew femtolitres, and at very low cost should be of enormous utility inthe generation of novel drug leads.

SUMMARY OF THE INVENTION

The present inventors have found that the powerful technique ofmicrofluidic control of microcapsules, in particular the electroniccontrol of microfluid based technology can be applied to thecompartmentalised microcapsule system described in Griffith & Tawfik(1998) which is herein incorporated by reference. The result is a novelmethod which is capable of creating and screening vast quantities ofcompounds both quickly and efficiently.

Thus, the invention, in a first aspect, provides a method for preparinga repertoire of compounds comprising the steps of:

(a) compartmentalising two or more sets of primary compounds intomicrocapsules; such that a proportion of the microcapsules containsmultiple copies of one or more compounds representative of each of saidsets, and wherein said one or more compounds represents a subset of theset of primary compounds; and

(b) forming secondary compounds in the microcapsules by chemicalreactions between primary compounds from different sets; wherein eitherone or both of steps (a) and (b) is performed under the microfluidiccontrol of fluidic species.

A compound is “representative” of a set where is member of said set;advantageously, therefore, each microcapsule contains compound(s) fromeach set. Although a microcapsule may contain more than one differentcompound from each set, it contains only a proportion of said set—i.e. asubset. The subset of a set advantageously represents no more that 10%of the members of the set; preferably, this figure is 9%, 8%, 7%, 6%,5%, 4%, 3%, 2%, 1% or less.

Most advantageously a microcapsule contains only a single compound fromeach set of primary compounds.

The sets of primary compounds used in the method of the invention canconsist of any number of different compounds. At least a first setcomprises two or more compounds; but the other set may be a singlecompound. Preferably, if a first set is a single compound, at least onefurther set comprises a repertoire of compounds. The larger thisrepertoire, the greater the number of different secondary compounds thatwill be generated.

Preferably, at least one set of compounds comprises a repertoire ofdifferent compounds. At least one set, however, may consist of a singlecompound, such that secondary compounds are all constructed based on orcontaining the single compound used in one set. The greater the numberof sets, and the greater the diversity of each set, the greater thefinal diversity of the secondary compounds generated.

Advantageously, in step (a) the number of different compounds percompartment will be equivalent to the number of primary compoundsforming the secondary compound in step (b).

In a second aspect, the invention provides a method for identifyingprimary compounds which react together to form secondary compoundscapable of binding to or modulating the activity of a target, comprisingthe steps of:

(a) compartmentalising two or more sets of primary compounds intomicrocapsules; such that a proportion of the microcapsules contains twoor more compounds;

(b) forming secondary compounds in the microcapsules by chemicalreactions between primary compounds from different sets; and

(c) identifying subsets of primary compounds which react to formsecondary compounds which bind to or modulate the activity of thetarget; wherein one or more of steps (a), (b) and (c) is performed underthe microfluidic control of fluidic species.

In a third aspect, the invention provides a method for synthesisingcompounds with enhanced ability to bind to or modulate the activity ofthe target, comprising the steps of:

(a) compartmentalising into microcapsules subsets of primary compoundsidentified in step (c) of the second aspect of the invention and,optionally, compartmentalising additional sets of primary compounds;

(b) forming secondary compounds in the microcapsules by chemicalreactions between primary compounds from different sets; and

(c) identifying subsets of primary compounds which react to formsecondary compounds which bind to or modulate the activity of thetarget; wherein one or more of steps (a), (b) and (c) is performed underthe microfluidic control of fluidic species.

Advantageously, steps (a) to (c) can be repeated, but after the firstcycle, step (a) comprises compartmentalising subsets of primarycompounds identified in step (c) into microcapsules and, optionally,compartmentalising additional sets of compounds.

In a fourth aspect, the invention provides a method for identifyingindividual compounds which bind to or modulate the activity of thetarget, comprising the steps of:

(a) compartmentalising into microcapsules a primary compound identifiedin step (c) of the second or third aspect of the invention andadditional sets of primary compounds;

(b) forming secondary compounds in the microcapsules by chemicalreactions between primary compounds from different sets; and

(c) identifying subsets of primary compounds which react to formsecondary compounds which bind to or modulate the activity of thetarget; wherein one or more of steps (a), (b) and (c) is performed underthe microfluidic control of fluidic species.

If the secondary compound is formed by the chemical reaction betweenmore than two primary compounds it can be identified by iterativelyrepeating steps (a) to (c), but after the first cycle, step (c)comprises compartmentalising the primary compound identified in step (c)of the second or third aspect of the invention, a primary compoundidentified in step (c) of each of the previous cycles (of the fourthaspect of the invention) and additional sets of primary molecules.

Preferably, the desired activity is selected from the group consistingof a binding activity and the modulation of the activity of a target.Advantageously, the target is compartmentalised into microcapsulestogether with the compounds.

Sets of compounds may be compartmentalised in different ways to achieveencapsulation of multiple copies of two or more compounds intomicrocapsules.

For example, small aliquots of an aqueous solution of each compound canbe deposited into an oil phase (advantageously containing surfactantsand/or other stabilising molecules) whilst applying mechanical energy,thereby dispersing each compound into multiple aqueous microcapsules,each of which contains (for the most part) a single sort of compound butmultiple copies thereof. Advantageously, the compounds can be depositedinto the oil phase in the form of droplets generated using inkjetprinting technology (Calvert, 2001; de Gans et al., 2004), and moreadvantageously by piezoelectric drop-on-demand (DOD) inkjet printingtechnology. Inkjet printing technology can be used to mix primarycompounds and, optionally, other reagents (e.g the target and reagentsto assay target activity) immediately prior to forming the emulsion.Advantageously, multiple compounds can be mixed with multiple targets ina combinatorial manner.

Thus, step (a) above can be modified such that it comprises formingseparate emulsion compartments containing two or more compounds andmixing the emulsion compartments to form an emulsified compoundrepertoire wherein a subset of the repertoire is represented in multiplecopies in any one microcapsule.

Moreover, compound libraries can be compartmentalised in highlymonodisperse microcapsules produced using microfluidic techniques. Forexample, aliquots of each compound can be compartmentalised into one ormore aqueous microcapsules (with less than 1.5% polydispersity) inwater-in-oil emulsions created by droplet break off in a co-flowingsteam of oil (Umbanhowar et al., 2000). Advantageously, the aqueousmicrocapsules are then transported by laminar-flow in a stream of oil inmicrofluidic channels (Thorsen et al., 2001). The microcapsulescontaining single compounds can, optionally, be split into two or moresmaller microcapsules using microfluidics (Link et al., 2004; Song etal., 2003). Microcapsules containing primary compounds can be fused withother microcapsules (Song et al., 2003) to form secondary compounds.

Microcapsules containing compounds can also, optionally, be fused withmicrocapsules containing a target. A single microcapsule containing atarget can, optionally, be split into two or more smaller microcapsuleswhich can subsequently be fused with microcapsules containing differentcompounds, or compounds at different concentrations. Advantageously, acompound and a target can be mixed by microcapsule fusion prior to asecond microcapsule fusion which delivers the reagents necessary toassay the activity of the target (e.g. the substrate for the target ifthe target is an enzyme). This allows time for the compound to bind tothe target. The microcapsules can be analysed and, optionally, sortedusing microfluidic devices (Fu et al., 2002).

In a further aspect, there is provided a method for preparing arepertoire of compounds comprising the steps of:

(a) attaching two or more sets of primary compounds onto microbeads;

(b) compartmentalising the microbeads into microcapsules such that aproportion of the microcapsules contains two or more microbeads;

(c) releasing at least one of the sets of primary compounds from themicrobeads;

(d) forming secondary compounds in the microcapsules by chemicalreactions between primary compounds from different sets; wherein one ormore of steps (a), (b), (c) and (d) is performed under the microfluidiccontrol of fluidic species.

Advantageously, the compounds are cleavable from the beads. Where morethan two sets of compounds are used, all the sets with the exception ofone are cleavable; preferably, they are all cleavable. The compounds maybe attached to the microbeads by photochemically cleavable linkers.

In a still further aspect, the invention provides a method foridentifying primary compounds which react together to form secondarycompounds capable of binding to or modulating the activity of a target,comprising the steps of:

(a) attaching two or more sets of primary compounds onto microbeads;

(b) compartmentalising the microbeads into microcapsules together withthe target such that many compartments contain two or more microbeads;

(c) releasing the primary compounds from the microbeads;

(d) forming secondary compounds in the microcapsules by chemicalreactions between primary compounds from different sets; and

(e) identifying subsets of primary compounds which react to formsecondary compounds which bind to or modulate the activity of thetarget; wherein one or more of steps (a), (b), (c), (d) and (e) isperformed under the microfluidic control of fluidic species.

Advantageously, in step (b) the modal number of microbeads percompartment will be equivalent to the number of primary compoundsforming the secondary compound in step (d).

In a further aspect, the invention provides a method for synthesisingcompounds with enhanced ability to bind to or modulate the activity ofthe target, comprising the steps of:

(a) attaching onto microbeads subsets of primary compounds identified instep (e) of the second aspect of the invention and, optionally,attaching additional sets of primary compounds;

(b) compartmentalising the microbeads into microcapsules together withthe target such that many compartments contain two or more microbeads;

(c) releasing the primary compounds from the microbeads;

(d) forming secondary compounds in the microcapsules by chemicalreactions between primary compounds from different sets; and

(e) identifying subsets of primary compounds which react to formsecondary compounds which bind to or modulate the activity of thetarget; wherein one or more of steps (a), (b), (c), (d) and (e) isperformed under the microfluidic control of fluidic species.

Advantageously, steps (a) to (e) can be repeated, but after the firstcycle, step (a) comprises attaching onto microbeads subsets of primarycompounds identified in step (e) and, optionally, attaching additionalsets of compounds.

In a further aspect, the invention provides a method for identifyingindividual compounds which bind to or modulate the activity of thetarget, comprising the steps of:

(a) attaching onto microbeads a primary compound identified in step (e)of the second or third aspect of the invention and additional sets ofprimary compounds;

(b) compartmentalising the microbeads into microcapsules together withthe target such that many compartments contain two or more microbeads;

(c) releasing the primary compounds from the microbeads;

(d) forming secondary compounds in the microcapsules by chemicalreactions between primary compounds from different sets; and

(e) identifying subsets of primary compounds which react to formsecondary compounds which bind to or modulate the activity of thetarget; wherein one or more of steps (a), (b), (c), (d) and (e) isperformed under the microfluidic control of fluidic species.

If the secondary compound is formed by the chemical reaction betweenmore than two primary compounds it can be identified by iterativelyrepeating steps (a) to (e), but after the first cycle, step (a)comprises attaching onto microbeads the primary compound identified instep (e) of the second or third aspect of the invention, a primarycompound identified in step (e) of each of the previous cycles (of thefourth aspect of the invention) and additional sets of primarymolecules.

Preferably, the desired activity is selected from the group consistingof a binding activity and the modulation of the activity of a target.Advantageously, the target is compartmentalised into microcapsulestogether with the microbeads.

According to a preferred implementation of the present invention, thecompounds may be screened according to an activity of the compound orderivative thereof which makes the microcapsule detectable as a whole.Accordingly, the invention provides a method wherein a compound with thedesired activity induces a change in the microcapsule, or a modificationof one or more molecules within the microcapsule, which enables themicrocapsule containing the compound and, optionally, the microbeadcarrying it to be identified. In this embodiment, therefore, themicrocapsules are either: (a) physically sorted from each otheraccording to the activity of the compound(s) contained therein, by forexample, placing an electric chrage on the microcapsule and ‘steering’the microcapsule using an electric field, and the contents of the sortedmicrocapsules analysed to determine the identity of the compound(s)which they contain; or (b) analysed directly without sorting todetermine the identity of the compound(s) which the microcapsulescontain.

According to a preferred embodiment of the present invention, thescreening of compounds may be performed by, for example:

(I) In a first embodiment, the microcapsules are screened according toan activity of the compound or derivative thereof which makes themicrocapsule detectable as a whole. Accordingly, the invention providesa method wherein a compound with the desired activity induces a changein the microcapsule, or a modification of one or more molecules withinthe microcapsule, which enables the microcapsule containing the compoundand the microbead carrying it to be identified. In this embodiment,therefore, the microcapsules are either: (a) physically sorted from eachother according to the activity of the compound(s) contained therein,the contents of the sorted microcapsules optionally pooled into one ormore common compartments, and the microcapsule contents analysed todetermine the identity of the compound(s); or (b) analysed directlywithout sorting to determine the identity of the compound(s) which themicrocapsules contained. Where the microcapsule contains microbeads, themicrobeads can be analysed to determine the compounds with which theyare coated.

(II) In a second embodiment, microbeads are analysed following poolingof the microcapsules into one or more common compartments. In thisembodiment, a compound having the desired activity modifies themicrobead which carried it (and which resides in the same microcapsule)in such a way as to make it identifiable in a subsequent step. Thereactions are stopped and the microcapsules are then broken so that 10′all the contents of the individual microcapsules are pooled. Modifiedmicrobeads are identified and either: (a) physically sorted from eachother according to the activity of the compound(s) coated on themicrobeads, and the sorted microbeads analysed to determine the identityof the compound(s) with which they are/were coated; or (b) analyseddirectly without sorting to determine the identity of the compound(s)with which the microbeads are/were coated. It is to be understood, ofcourse, that modification of the microbead may be direct, in that it iscaused by the direct action of the compound, or indirect, in which aseries of reactions, one or more of which involve the compound havingthe desired activity, leads to modification of the microbead.Advantageously, the target is bound to the microbead and is a ligand andthe compound within the microcapsule binds, directly or indirectly, tosaid ligand to enable the isolation of the microbead. In anotherconfiguration, a substrate for the target is and is bound to themicrobead, and the activity of the compound within the microcapsuleresults, directly or indirectly, in the conversion of said substrateinto a product which remains part of the microbead and enables itsisolation. Alternatively, the activity of the compound may prevent orinhibit the conversion of said substrate into product. Moreover, theproduct of the activity of the compound within the microcapsule canresult, directly or indirectly, in the generation of a product which issubsequently complexed with the microbead and enables itsidentification.

(III) In a third embodiment, the microbeads are analysed followingpooling of the microcapsules into one or more common compartments. Inthis embodiment, a compound with a desired activity induces a change inthe microcapsule containing the compound and the microbead which carriesit. This change, when detected, triggers the modification of themicrobead within the compartment. The reactions are stopped and themicrocapsules are then broken so that all the contents of the individualmicrocapsules are pooled. Modified microbeads are identified and either:(a) physically sorted from each other according to the activity of thecompound(s) coated on the microbeads, and the sorted microbeads analysedto determine the identity of the compound(s) with which they are/werecoated; or (b) analysed directly without sorting to determine theidentity of the compound(s) with which the microbeads are/were coated.

The microcapsules or microbeads may be modified by the action of thecompound(s) such as to change their optical properties and/or electricalcharge properties. For example, the modification of the microbead canenable it to be further modified outside the microcapsule so as toinduce a change in its optical and/or electrical charge properties.

In another embodiment, the change in optical and/or electrical chargeproperties of the microcapsules or microbeads is due to binding of acompound with distinctive optical and/or electrical charge propertiesrespectively to the target.

Moreover, the change in optical and/or electrical charge properties ofthe microcapsules or microbeads can be due to binding of a target withdistinctive optical and/or electrical charge properties respectively bythe compound.

The change in the optical and/or electrical charge properties of themicrocapsule may be due to modulation of the activity of the target bythe compound. The compound may activate or inhibit the activity of thetarget. For example, if the target is an enzyme, the substrate and theproduct of the reaction catalysed by the target can have differentoptical and/or electrical charge properties. Advantageously, thesubstrate and product have different fluorescence properties. In thecase where the microcapsules contain microbeads, both the substrate andthe product can have similar optical and/or electrical chargeproperties, but only the product of the reaction, and not the substrate,binds to, or reacts with, the microbead, thereby changing the opticaland/or electrical charge properties of the microbead.

The change in optical and/or electrical charge properties of themicrocapsules or microbeads can also be due to the different opticaland/or electrical charge properties of the target and the product of thereaction being selected. Where both target and product have similaroptical and/or electrical charge properties, only the product of thereaction being selected, and not the target, binds to, or reacts with,the microbead, thereby changing the optical and/or electrical chargeproperties of the microcapsules or microbeads.

In a further configuration, further reagents specifically bind to, orspecifically react with, the product (and not the substrate) attached toor contained in the microcapsule or microbead, thereby altering theoptical and/or electrical charge properties of the microcapsule ormicrobead.

Advantageously, microbeads modified directly or indirectly by theactivity of the compound are further modified by Tyramide SignalAmplification (TSA™; NEN), resulting directly or indirectly in a changein the optical properties of said microcapsules or microbeads therebyenabling their separation.

It is to be understood that the detected change in the compartment maybe caused by the direct action of the compound, or indirect action, inwhich a series of reactions, one or more of which involve the compoundhaving the desired activity leads to the detected change.

Where the compounds are attached to beads, the density with whichcompounds are coated onto the microbeads, combined with the size of themicrocapsule will determine the concentration of the compound in themicrocapsule. High compound coating densities and small microcapsuleswill both give higher compound concentrations which may be advantageousfor the selection of molecules with a low affinity for the target.Conversely, low compound coating densities and large microcapsules willboth give lower compound concentrations which may be advantageous forthe selection of molecules with a high affinity for the target.

Preferably, microencapsulation is achieved by forming an emulsion.

The microbead can be nonmagnetic, magnetic or paramagnetic.

Repertoires of compounds can be encapsulated so as to have multiplecopies of a single compound in a microcapsule in different ways. Forexample, thin tubes connected to the microfluidic device can be dippedinto reservoirs containing the desired compounds, and capillary actioncan be used to draw the desired compound from the reservoir into themicrofluidic device. This method allows the microfluidic device to beloaded with compounds prepared outside the device.

Moreover, compound libraries can be compartmentalised in highlymonodisperse microcapsules produced using microfluidic techniques. Forexample, aliquots of each compound can be compartmentalised into one ormore aqueous microcapsules (with less than 1.5% polydispersity) inwater-in-oil emulsions created by droplet break off in a co-flowingsteam of oil (Umbanhowar et al., 2000). Advantageously, the aqueousmicrocapsules are then transported by laminar-flow in a stream of oil inmicrofluidic channels (Thorsen et al., 2001). These microcapsulescontaining single compounds can, optionally, be split into two or moresmaller microcapsules using microfluidics (Link et al., 2004; Song etal., 2003). The microcapsules containing single compounds can,optionally be fused with other microcapsules (Song et al., 2003)containing a target. A single microcapsule containing a target can,optionally, be split into two or more smaller microcapsules which cansubsequently be fused with microcapsules containing different compounds,or compounds at different concentrations. Advantageously, a compound anda target can be mixed by microcapsule fusion, prior to a secondmicrocapsule fusion which delivers the necessary to assay the activityof the target (e.g. the substrate for the target if the target is anenzyme). This allows time for the compound to bind to the target. Themicrocapsules can be analysed and, optionally, sorted using microfluidicdevices (Fu et al., 2002).

Methods of controlling and manipulating of fluidic species are alsodescribed, for example, in U.S. Provisional Patent Application Ser. No.60/498,091, filed Aug. 27, 2003, by Link, et. al.; U.S. ProvisionalPatent Application Ser. No. 60/392,195, filed Jun. 28, 2002, by Stone,et. al.; U.S. Provisional Patent Application Ser. No. 60/424,042, filedNov. 5, 2002, by Link, et al.; U.S. Pat. No. 5,512,131, issued Apr. 30,1996 to Kumar, et al.; International Patent Publication WO 96/29629,published Jun. 26, 1996 by Whitesides, et al.; U.S. Pat. No. 6,355,198,issued Mar. 12, 2002 to Kim, et al.; International Patent ApplicationSerial No.: PCT/US01/16973, filed May 25, 2001 by Anderson, et al.,published as WO 01/89787 on Nov. 29, 2001; International PatentApplication Serial No. PCT/US03/20542, filed Jun. 30, 2003 by Stone, etal., published as WO 2004/002627 on Jan. 8, 2004; International PatentApplication Serial No. PCT/US2004/010903, filed Apr. 9, 2004 by Link, etal.; and U.S. Provisional Patent Application Ser. No. 60/461,954, filedApr. 10, 2003, by Link, et al.; each of which is incorporated herein byreference.

In various aspects of the invention, a fluidic system as disclosedherein may include a droplet formation system, a droplet fusing system,a droplet splitting system, a sensing system, a controller, and/or adroplet sorting and/or separation system, or any combination of thesesystems. Such systems and methods may be positioned in any suitableorder, depending on a particular application, and in some cases,multiple systems of a given type may be used, for example, two or moredroplet formation systems, two or more droplet separation systems, etc.As examples of arrangements, systems of the invention can be arranged toform droplets, to dilute fluids, to control the concentration of specieswithin droplets, to sort droplets to select those with a desiredconcentration of species or entities (e.g., droplets each containing onemolecule of reactant), to fuse individual droplets to cause reactionbetween species contained in the individual droplets, to determinereaction(s) and/or rates of reaction(s) in one or more droplets, etc.Many other arrangements can be practiced in accordance with theinvention.

One aspect of the invention relates to systems and methods for producingdroplets of a first liquid surrounded by a second liquid 2. The firstand second liquids may be essentially immiscible in many cases, i.e.,immiscible on a time scale of interest (e.g., the time it takes afluidic droplet to be transported through a particular system ordevice). In certain cases, the droplets may each be substantially thesame shape or size, as further described below. The first liquid mayalso contain other species, for example, certain molecular species(e.g., as further discussed below), cells, particles, etc.

In one set of embodiments, electric charge may be created on a firstliquid surrounded by a second liquid, which may cause the first liquidto separate into individual droplets within the second liquid. In someembodiments, the first liquid and the second liquid may be present in achannel, e.g., a microfluidic channel, or other constricted space thatfacilitates application of an electric field to the first liquid (whichmay be “AC” or alternating current, “DC” or direct current etc.), forexample, by limiting movement of the first liquid with respect to thesecond liquid. Thus, the first liquid can be present as a series ofindividual charged and/or electrically inducible droplets within thesecond liquid. In one embodiment, the electric force exerted on thefluidic droplet may be large enough to cause the droplet to move withinthe second liquid. In some cases, the electric force exerted on thefluidic droplet may be used to direct a desired motion of the dropletwithin the second liquid, for example, to or within a channel or amicrofluidic channel.

Electric charge may be created in the first liquid within the secondliquid using any suitable technique, for example, by placing the firstliquid within an electric field (which may be AC, DC, etc.), and/orcausing a reaction to occur that causes the first liquid to have anelectric charge, for example, a chemical reaction, an ionic reaction, aphotocatalyzed reaction, etc. In one embodiment, the first liquid is anelectrical conductor. As used herein, a “conductor” is a material havinga conductivity of at least about the conductivity of 18 megohm (MOhm orMΩ) water. The second liquid surrounding the first liquid may have aconductivity less than that of the first liquid. For instance, thesecond liquid may be an insulator, relative to the first liquid, or atleast a “leaky insulator,” i.e., the second liquid is able to at leastpartially electrically insulate the first liquid for at least a shortperiod of time. Those of ordinary skill in the art will be able toidentify the conductivity of fluids. In one non-limiting embodiment, thefirst liquid may be substantially hydrophilic, and the second liquidsurrounding the first liquid may be substantially hydrophobic. In analternative embodiment, the microcapsules or microbeads are analysed bydetection of a change in their fluorescence. For example, microbeads canbe analysed by flow cytometry and, optionally sorted using afluorescence activated cell sorter (FACS). The different fluorescenceproperties of the target and the product can be due to fluorescenceresonance energy transfer (FRET).

In a further embodiment, the internal environment of the microcapsulescan be modified by the addition of one or more reagents to thecontinuous phase of the emulsion. This allows reagents to be diffused into the microcapsules during the reaction, if necessary.

The invention moreover relates to a method according to the precedingaspects, further comprising the step of isolating the secondary compoundproduced by reaction of the primary compounds and optionally furthercomprising the step of manufacturing one or more secondary compounds.

The invention also provides for a product when identified according tothe invention. As used in this context, a “product” may refer to anycompound, selectable according to the invention.

Further embodiments of the invention are described in the detaileddescription below and in the accompanying claims.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B illustrate the splitting of droplets in accordance withone embodiment of the invention;

FIGS. 2A and 2B illustrate an apparatus in accordance with an embodimentof the invention, before the application of an electric field thereto;

FIGS. 3A and 3B illustrate the apparatus of FIGS. 2A and 2B after theapplication of an electric field thereto;

FIGS. 4A and 4B illustrate the apparatus of FIGS. 2A and 2B after theapplication of a reversed electric field thereto;

FIG. 5 is a schematic diagram of droplet splitting, in accordance withone embodiment of the invention;

FIGS. 6A and 6B are schematic diagrams of additional embodiments of theinvention;

FIGS. 7 a and 7 b are schematic diagrams of the formation ofmicrofluidic droplets in accordance with the present invention;

FIGS. 8 a-f illustrate the splitting of droplets in accordance with theinvention;

FIGS. 9 a-d illustrate the induction of dipoles in droplets inaccordance with the invention;

FIGS. 10 a-d illustrate the sorting of microcasules by altering the flowof carrier fluid in a microfluidic system;

FIGS. 11 a-c illustrate the use of pressure changes in the microfluidicsystem to control the direction of flow of droplets;

FIGS. 12 a-j illustrate flow patterns for droplets in microfluidicsystems in accordance with the invention;

FIGS. 13 a-d illustrate the use of oppositely charged droplets in theinvention;

FIGS. 14 a, 14 b and 14 c are illustrations of the formation andmaintenance of microfluidic droplets using three immiscible liquids;

FIG. 15. Compound screening using microdroplets in a microfluidicsystem. Panel A: schematic of the core system. Panel B: process blockdiagram showing the modules in the core system. Microdroplets containinga target enzyme are fused with microdroplets each of which contain adifferent compound from a compound library. After allowing time for thecompounds to bind to the target enzyme each microdroplet is fused withanother microdroplet containing a fluorogenic enzyme substrate. The rateof the enzymatic reaction is determined by measuring the fluorescence ofeach microdroplet, ideally at multiple points (corresponding todifferent times). Microdroplets containing compounds with desiredactivities can, if required, be sorted and collected.

FIG. 16. Examples of microdroplet formation and manipulation usingmicrofluidics. Panel A: microdroplets can be created at up to 10⁴ sec⁻¹by hydrodynamic-focussing (top two panels) and show <1.5% polydispersity(bottom panel). Panel B: microdroplets can be split symmetrically orasymmetrically. Panel C: microdroplets carrying positive (+q) andnegative (−q) electrical charges fuse spontaneously. Panel D: chargedmicrodroplets can also be steered using an applied electrical field (E).

FIG. 17. Examples of PTP1B inhibitors. Compounds with abis-difluoromethylene phosphonate moiety (e.g. 2) have significantlymore potency than those with a single moiety (e.g. 1).

FIG. 18. Screening PTP1B inhibitors using microencapsulation.Polystyrene beads with surface carboxylate groups, died with orange orred fluorochromes (Fulton et al., 1997), are derivatised with aphosphopeptide PTP1B substrate, and either PTP1B inhibitors ornon-inhibitory compounds, attached via a cleavable linker (1). Aftermixing the beads, single beads and target enzyme (PTP1B) are colocalisedin a microcompartment by forming a water-in-oil emulsion using amicrofluidic device (2). The compound is released (photochemically) (3).Inhibitors reduce the amount of substrate converted to product(dephosphorylated peptide) (4). The enzyme reaction is stopped and theemulsion is broken (5). After labelling with green fluorescentanti-substrate antibodies, beads are analysed by 3-colour flow cytometryto simultaneously determine extent of inhibition and the compound on thebeads (6). Compound libraries can be coupled to optically tagged beads(see below) and rapidly decoded by flow cytometry (at up to 100,000beads s⁻¹). Hit compounds are re-synthesised for furthercharacterisation (7) or elaborated and rescreened in a process ofsynthetic evolution (8).

FIG. 19. Synthesising PTP1B inhibitors in an emulsion. Two types ofbeads are created, differentially labelled with orange and redfluorochromes, and derivatised with two types of molecule, A or B(neither, one, or both of which contain a difluoromethylene phosphonatemoiety), attached via a reversible connection (a Schiff base). Beads areemulsified using a microfluidic device to give, on average, two beadsper compartment. The molecules, A & B, are released from the beads inthe compartment and react to form a new molecule, A-B, (in solution). IfA-B is a PTP1B inhibitor the PTP1B substrate also on the beads is notdephosphorylated and these beads identified by flow cytometry as FIG.18.

FIG. 20. Small molecule evolution using four-component reactions. Foursets of 25 beads are created, each derivatised with one of 25 variantsof molecules A, B, C or D, emulsified to give, on average, 4 beads percompartment, the compounds released to synthesise a large combinatorialrepertoire (4×10⁵) in situ and screened as FIG. 18. Low affinityinhibitors will be ‘recombined’ by re-screening mixtures of beadscarrying moieties identified in inhibitors. Beads carrying a moietyfound in inhibitors (e.g. A₁₀) can also be mixed with complete sets ofbeads coated with B, C and D and screened. If a moiety (say B₈) is thenidentified as a component of an inhibitor, beads coated with A₁₀ and B₈can be mixed with complete sets of beads C and D and the processrepeated. This process of ‘mutation’ also results in deconvolution.After fixing three of the four moieties in active compounds,deconvolution can be completed using multiplex bead analysis as above.Compounds can be re-diversified or ‘mutated’ using bead sets carryingvariant, exploded sets of the molecules used in the original libraries.

FIG. 21. Compartmentalisation of small molecules inwater-in-fluorocarbon emulsions. Water-in-perfluorooctyl bromideemulsions were made containing texas red (1 mM) and calcein (1 mM) inthe aqueous phase by homogenisation as described in example 6 The twoemulsions were mixed by vortexing and imaged by epifluorescencemicroscopy after 24 hours. No exchange of texas-red (red fluorescence)and calcein (green fluorescence) between microdroplets could beobserved.

FIG. 22. Primary compounds for the synthesis of PTP1B inhibitors. Anamine (A) and an aldehyde (B) with difluoromethylene phosphonatemoieties. Amine A reacts with aldehyde B in the aqueous microcapsules ofa water-in-oil emulsion to generate the imine C which is a potent PTP1Binhibitor. C can be reduced in situ using cyanoborohydride to generatethe stable, amine D.

FIG. 23 Charged droplet generation. (A), Oil and water streams convergeat a 30 micron orifice. A voltage V applied to indium-tin-oxide (ITO)electrodes on the glass produces an electric field E to capacitivelycharges the aqueous-oil interface. Drop size is independent of charge atlow field strengths but decreases at higher fields, as shown in thephotomicrographs, [(B) V=0, (C) V=400, (D) V=600 and (E) V=800] athigher fields. (F) Droplet size as a function of voltage showing thecrossover between flow-dominated and field-dominated snap-off for threedifferent flow rates of the continuous phase oil (Q_(c)=80 nL/s, 110nL/s, and 140 nL/s). The infusion rate of the water is constant Q_(d)=20nL/s.}

FIG. 24 Coalescing drops. (A) Drops having opposite sign ofelectrostatic charge can be generated by applying a voltage across thetwo aqueous streams. (B) In the absence of the field the frequency andtiming of drop formation at the two nozzles are independent and eachnozzle produces a different size drop at a different frequency; infusionrates are the same at both nozzles. After the confluence of the twostreams, drops from the upper and lower nozzles stay in their respectivehalves of the stream and due to surfactant there are no coalescenceevents even in the case of large slugs that fill the channel width. (C)With an applied voltage of 200V across the 500 micron separation of thenozzles, the drops simultaneously break-off from the two nozzles and areidentical; simultaneous drop formation can be achieved for unequalinfusion rates of the aqueous streams even up to a factor of twodifference in volumes. (D) The fraction of the drops that encounter eachother and coalesce increases linearly above a critical field when asurfactant, sorbiton-monooleate 3% is present.

FIGS. 25 a and 25 b: Droplets carrying a pH sensitive dye coalesce withdroplets of a different pH fluid. Chaotic advection rapidly mixes thetwo fluids through a combination of translation and rotation as thedroplets pass around corners.

FIG. 26: Diffusion limited and rapid mixing strategies. (A) Drops meetand coalesce along the direction of E and then move off in aperpendicular direction, as sketched the counter rotating vortices aftercoalescence do not mix the two fluid parts as each vortex contains asingle material. (B) As the drops approach each other the increasingfield causes there interfaces to deform and (C) a bridge to jump outconnecting the drops, to create (D) in the case of 20 nm silicaparticles and MgCl_(—)2 a sharp interface where the particles begin togel. (E) A typical unmixed droplet with particles in one hemisphere. (F)To achieve fast mixing, droplets are brought together in the directionperpendicular to the electric field and move off in the directionparallel to the direction they merged along. Counter rotating vortexesare then created where each vortex is composed of half of the contentesfrom each of the premerger-droplets. (G) Shows a pH sensitive dye in thelower drop and a different, pH fluid in the upper droplet. (H) Aftermerger the droplets are split by a sharp line. (I) A uniform intensityindicating that mixing has been occurred is achieved in the dropletafter it translates one diameter, typically this takes 1 to 2 ms.

FIG. 27 Time delay reaction module. (A) Droplets of perfluorodecalinealternate with aqueous droplets in a hexadecane carrier fluid. The‘single-file’ ordering of the droplets provides for long delays withessentially no deviation in the precise spacing of aqueous droplets ordroplet order. (B) Increasing the width and height of the channel tocreate a ‘large cross-sectional area’ channel, provides for extremelylong time delays from minutes to hours. The exact ordering and spacingbetween the droplets is not maintained in this type of delay line.

FIG. 28 Recharging neutral drops. (A) Schematic to recharge neutraldrops by breaking them in the presence of an electric field. Unchargeddrops (q=0) are polarized in an electric field (E_(S)≠0), and providedE_(S) is sufficiently large, as shown in the photomicrograph of (B),they break into two oppositely charged daughter drops in the extensionalflow at a bifurcation. The enlargement of the dashed rectangle, shown in(C), reveals that the charged drops are stretched in the electric fieldE_(S) but return to spherical on contacting the electrodes indicated bydashed vertical lines.

FIG. 29 Detection module. One or more lasers are coupled to an opticalfibre that is used to excite the fluorescence in each droplet as itpasses over the fibre. The fluorescence is collected by the same fibreand dichroic beam splitters separate off specific wavelengths of thefluorescent light and the intensity of the fluorescence is measured witha photomultiplier tube (PMT) after the light passes through a band-passfilter.

FIG. 30 Manipulating charged drops. In (A) charged drops alternatelyenter the right and left channels when there is no field applied(E_(S)=0). The sketch in (B) shows the layout for using an electricfield E_(S) to select the channel charged drops will enter at abifurcation. When an electric field is applied to the right (C), thedrops enter the right branch at the bifurcation; they enter the leftbranch when the field is reversed (D). After the bifurcation, thedistance between drops is reduced to half what it was before indicatingthe oil stream is evenly divided. The inset of (D) shows the deformationin the shape of a highly charged drop in an electric field.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The term “microcapsule” is used herein in accordance with the meaningnormally assigned thereto in the art and further described hereinbelow.In essence, however, a microcapsule is an artificial compartment whosedelimiting borders restrict the exchange of the components of themolecular mechanisms described herein which allow the identification ofthe molecule with the desired activity. The delimiting borderspreferably completely enclose the contents of the microcapsule.Preferably, the microcapsules used in the method of the presentinvention will be capable of being produced in very large numbers, andthereby to compartmentalise a library of compounds. Optionally, thecompounds can be attached to microbeads. The microcapsules used hereinallow mixing and sorting to be performed thereon, in order to facilitatethe high throughput potential of the methods of the invention.Microcapsules according to the present invention can be a droplet of onefluid in a different fluid, where the confined components are soluble inthe droplet but not in the carrier fluid, and in another embodimentthere is another material defining a wall, such as a membrane (e.g. inthe context of lipid vesicles; liposomes) or non-ionic surfactantvesicles, or those with rigid, nonpermeable membranes, or semipermeablemembranes. Arrays of liquid droplets on solid surfaces, multiwell platesand “plugs” in microfluidic systems, that is fluid droplets that are notcompletely surrounded by a second fluid as defined herein, are notmicrocapsules as defined herein.

A “proportion” of the microcapsules, which is defined as comprising twoor more compounds, or two or microbeads, is any fraction of themicrocapsules in question, including all of said microcapsules.Advantageously, it is at least 25% thereof, preferably 50%, and morepreferably 60%, 70%, 80%, 90% or 95%.

The term “microbead” is used herein in accordance with the meaningnormally assigned thereto in the art and further described hereinbelow.Microbeads, are also known by those skilled in the art as microspheres,latex particles, beads, or minibeads, are available in diameters from 20nm to 1 mm and can be made from a variety of materials including silicaand a variety of polymers, copolymers and terpolymers. Highly uniformderivatised and non-derivatised nonmagnetic and paramagneticmicroparticles (beads) are commercially available from many sources(e.g. Sigma, Bangs Laboratories, Luminex and Molecular Probes) (Fomusekand Vetvicka, 1986).

Microbeads can be “compartmentalised” in accordance with the presentinvention by distribution into microcapsules. For example, in apreferred aspect the microbeads can be placed in a water/oil mixture andemulsified to form a water-in-oil emulsion comprising microcapsulesaccording to the invention. The concentration of the microbeads can beadjusted to control the number of microbeads, which on average, appearin each microcapsule. Advantageously, the concentration of themicrobeads can be adjusted such that, on average a single microbeadappears in only 10-20% of the microcapsules, thus assuring that thereare very few microcapsules with more than one microbead.

The term “compound” is used herein in accordance with the meaningnormally assigned thereto in the art. The term compound is used in itsbroadest sense i.e. a substance comprising two or more elements in fixedproportions, including molecules and supramolecular complexes. Thisdefinition includes small molecules (typically <500 Daltons) which makeup the majority of pharmaceuticals. However, the definition alsoincludes larger molecules, including polymers, for example polypeptides,nucleic acids and carbohydrates, and supramolecular complexes thereof.

The term “primary compound” is used herein to indicate a compound whichis compartmentalised in a microcapsule or coupled to a bead.

The term “secondary compound” is used herein to indicate a compoundwhich is formed by the reaction between two or more primary compounds ina microcapsule, optionally after the release of at least one of theprimary molecules from a microbead.

Advantageously, all primary molecules are released from the microbeads.The secondary compound may be the result of a covalent or non-covalentreaction between the primary compounds.

The term “scaffold” is used, herein in accordance with the meaningnormally assigned thereto in the art. That is to say a core portion of amolecule common to all members of a combinatorial library (Maclean etal., 1999). Secondary compounds may optionally comprise scaffolds.

A “repertoire” of compounds is a group of diverse compounds, which mayalso be referred to as a library of compounds. Repertoires of compoundsmay be generated by any means known in the art, including combinatorialchemistry, compound evolution, or purchased from commercial sources suchas Sigma Aldrich, Discovery Partners International, Maybridge andTripos. A repertoire advantageously comprises at least 10², 10³, 10⁴,10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹ or more different compounds, whichmay be related or unrelated in structure or function.

A “set” of compounds may be a repertoire of compounds or any part of arepertoire, including a single compound species. The invention envisagesthe use of two or more sets of compounds, which are reacted together.The sets may be derived from a single repertoire, or a plurality ofdifferent repertoires.

Compounds can be “released” from a microbead by cleavage of a linkerwhich effects the attachment of the compound to the microbead. Releaseof the compounds from the microbead allows the compounds to interactmore freely with other contents of the microcapsule, and to be involvedin reactions therein and optionally to become combined with otherreagents to form new compounds, complexes, molecules or supramolecularcomplexes. Cleavage of linkers can be performed by any means, with meanssuch as photochemical cleavage which can be effected from without themicrocapsule being preferred. Photochemically cleavable linkers areknown in the art (see for example (Gordon and Balasubramanian, 1999))and further described below.

As used herein, the “target” is any compound, molecule, orsupramolecular complex. Typical targets include targets of medicalsignificance, including drug targets such as receptors, for example Gprotein coupled receptors and hormone receptors; transcription factors,protein kinases and phosphatases involved in signalling pathways; geneproducts specific to microorganisms, such as components of cell walls,replicases and other enzymes; industrially relevant targets, such asenzymes used in the food industry, reagents intended for research orproduction purposes, and the like.

An “activity”, as referred to herein in connection with the modulationof an activity of a target, can be any activity of the target or anactivity of a molecule which is influenced by the target, which ismodulatable directly or indirectly by a compound or compounds as assayedherein. The activity of the target may be any measurable biological orchemical activity, including binding activity, an enzymatic activity, anactivating or inhibitory activity on a third enzyme or other molecule,the ability to cause disease or influence metabolism or other functions,and the like. Activation and inhibition, as referred to herein, denotethe increase or decrease of a desired activity 1.5 fold, 2 fold, 3 fold,4 fold, 5 fold, 10 fold, 100 fold or more. Where the modulation isinactivation, the inactivation can be substantially completeinactivation. The desired activity may moreover be purely a bindingactivity, which may or may not involve the modulation of the activity ofthe target bound to.

A compound defined herein as “low molecular weight” or a “smallmolecule” is a molecule commonly referred to in the pharmaceutical artsas a “small molecule”. Such compounds are smaller than polypeptides andother, large molecular complexes and can be easily administered to andassimilated by patients and other subjects. Small molecule drugs canadvantageously be formulated for oral administration or intramuscularinjection. For example, a small molecule may have a molecular weight ofup to 2000 Dalton; preferably up to 1000 Dalton; advantageously between250 and 750 Dalton; and more preferably less than 500 Dalton.

A “selectable change” is any change which can be measured and acted uponto identify or isolate the compound which causes it. The selection maytake place at the level of the microcapsule, the microbead, or thecompound itself, optionally when complexed with another reagent. Aparticularly advantageous embodiment is optical detection, in which theselectable change is a change in optical properties, which can bedetected and acted upon for instance in a FACS device to separatemicrocapsules or microbeads displaying the desired change.

As used herein, a ‘change in optical properties’ refers to any change inabsorption or emission of electromagnetic radiation, including changesin absorbance, luminescence, phosphorescence or fluorescence. All suchproperties are included in the term “optical”. Microcapsules ormicrobeads can be identified and, optionally, sorted, for example, byluminescence, fluorescence or phosphorescence activated sorting. In apreferred embodiment, flow cytometry is employed to identify and,optionally, sort microcapsules or microbeads. A variety of opticalproperties can be used for analysis and to trigger sorting, includinglight scattering (Kerker, 1983) and fluorescence polarisation (Rollandet al., 1985). In a highly preferred embodiment microcapsules ormicrobeads are analysed and, optionally, sorted using a fluorescenceactivated cell sorter (FACS) (Norman, 1980; Mackenzie and Pinder, 1986).

The compounds in microcapsules or on beads can be identified using avariety of techniques familiar to those skilled in the art, includingmass spectroscopy, chemical tagging or optical tagging.

As used herein, “or” is understood to mean “inclusively or,” i.e., theinclusion of at least one, but including more than one, of a number orlist of elements. In contrast, the term “exclusively or” refers to theinclusion of exactly one element of a number or list of elements.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, should be understood to mean “at leastone.”

The term “about,” as used herein in reference to a numerical parameter(for example, a physical, chemical, electrical, or biological property),will be understood by those of ordinary skill in the art to be anapproximation of a numerical value, the exact value of which may besubject to errors such as those resulting from measurement errors of thenumerical parameter, uncertainties resulting from the variability and/orreproducibility of the numerical parameter (for example, in separateexperiments), and the like.

As used herein, a “cell” is given its ordinary meaning as used inbiology. The cell may be any cell or cell type. For example, the cellmay be a bacterium or other single-cell organism, a plant cell, or ananimal cell. If the cell is a single-cell organism, then the cell maybe, for example, a protozoan, a trypanosome, an amoeba, a yeast cell,algae, etc. If the cell is an animal cell, the cell may be, for example,an invertebrate cell (e.g., a cell from a fruit fly), a fish cell (e.g.,a zebrafish cell), an amphibian cell (e.g., a frog cell), a reptilecell, a bird cell, or a mammalian cell such as a primate cell, a bovinecell, a horse cell, a porcine cell, a goat cell, a dog cell, a cat cell,or a cell from a rodent such as a rat or a mouse. If the cell is from amulticellular organism, the cell may be from any part of the organism.For instance, if the cell is from an animal, the cell may be a cardiaccell, a fibroblast, a keratinocyte, a heptaocyte, a chondracyte, aneural cell, a osteocyte, a muscle cell, a blood cell, an endothelialcell, an immune cell (e.g., a T-cell, a B-cell, a macrophage, aneutrophil, a basophil, a mast cell, an eosinophil), a stem cell, etc.In some cases, the cell may be a genetically engineered cell. In certainembodiments, the cell may be a Chinese hamster ovarian (“CHO”) cell or a3T3 cell.

“Microfluidic,” as used herein, refers to a device, apparatus or systemincluding at least one fluid channel having a cross-sectional dimensionof less than 1 mm, and a ratio of length to largest cross-sectionaldimension of at least 3:1. A “microfluidic channel,” as used herein, isa channel meeting these criteria.

Accordingly the term ‘microfluidic control’ (of a system/method) asdescribed herein refers to a method/system which comprises the use ofdevice or apparatus including at least one fluid channel having across-sectional dimension of less than 1 mm, and a ratio of length tolargest cross-sectional dimension of at least 3:1.

Electronic microfluidic control (of a method/system) as referred toherein refers to a microfluidic method/system in which one or more stepsof the method/system involves the production of an electronic charge onat least, a proportion of the microcapsules used in the microfulidicmethod/system.

The “cross-sectional dimension” of the channel is measured perpendicularto the direction of fluid flow. Most fluid channels in components of theinvention have maximum cross-sectional dimensions less than 2 mm, and insome cases, less than 1 mm. In one set of embodiments, all fluidchannels containing embodiments of the invention are microfluidic orhave a largest cross sectional dimension of no more than 2 mm or 1 mm.In another embodiment, the fluid channels may be formed in part by asingle component (e.g. an etched substrate or molded unit). Of course,larger channels, tubes, chambers, reservoirs, etc. can be used to storefluids in bulk and to deliver fluids to components of the invention. Inone set of embodiments, the maximum cross-sectional dimension of thechannel(s) containing embodiments of the invention are less than 500microns, less than 200 microns, less than 100 microns, less than 50microns, or less than 25 microns.

A “channel,” as used herein, means a feature on or in an article(substrate) that at least partially directs the flow of a fluid. Thechannel can have any cross-sectional shape (circular, oval, triangular,irregular, square or rectangular, or the like) and can be covered oruncovered. In embodiments where it is completely covered, at least oneportion of the channel can have a cross-section that is completelyenclosed, or the entire channel may be completely enclosed along itsentire length with the exception of its inlet(s) and outlet(s). Achannel may also have an aspect ratio (length to average cross sectionaldimension) of at least 2:1, more typically at least 3:1, 5:1, or 10:1 ormore. An open channel generally will include characteristics thatfacilitate control over fluid transport, e.g., structuralcharacteristics (an elongated indentation) and/or physical or chemicalcharacteristics (hydrophobicity vs. hydrophilicity) or othercharacteristics that can exert a force (e.g., a containing force) on afluid. The fluid within the channel may partially or completely fill thechannel. In some cases where an open channel is used, the fluid may beheld within the channel, for example, using surface tension (i.e., aconcave or convex meniscus).

The channel may be of any size, for example, having a largest dimensionperpendicular to fluid flow of less than about 5 mm or 2 mm, or lessthan about 1 mm, or less than about 500 microns, less than about 200microns, less than about 100 microns, less than about 60 microns, lessthan about 50 microns, less than about 40 microns, less than about 30microns, less than about 25 microns, less than about 10 microns, lessthan about 3 microns, less than about 1 micron, less than about 300 nm,less than about 100 nm, less than about 30 nm, or less than about 10 nm.In some cases the dimensions of the channel may be chosen such thatfluid is able to freely flow through the article or substrate. Thedimensions of the channel may also be chosen, for example, to allow acertain volumetric or linear flowrate of fluid in the channel. Ofcourse, the number of channels and the shape of the channels can bevaried by any method known to those of ordinary skill in the art. Insome cases, more than one channel or capillary may be used. For example,two or more channels may be used, where they are positioned inside eachother, positioned adjacent to each other, positioned to intersect witheach other, etc.

As used herein, “integral” means that portions of components are joinedin such a way that they cannot be separated from each other withoutcutting or breaking the components from each other.

A “droplet,” as used herein is an isolated portion of a first fluid thatis completely surrounded by a second fluid. It is to be noted that adroplet is not necessarily spherical, but may assume other shapes aswell, for example, depending on the external environment. In oneembodiment, the droplet has a minimum cross-sectional dimension that issubstantially equal to the largest dimension of the channelperpendicular to fluid flow in which the droplet is located.

The “average diameter” of a population of droplets is the arithmeticaverage of the diameters of the droplets. Those of ordinary skill in theart will be able to determine the average diameter of a population ofdroplets, for example, using laser light scattering or other knowntechniques. The diameter of a droplet, in a non-spherical droplet, isthe mathematically-defined average diameter of the droplet, integratedacross the entire surface. As non-limiting examples, the averagediameter of a droplet may be less than about 1 mm, less than about 500micrometers, less than about 200 micrometers, less than about 100micrometers, less than about 75 micrometers, less than about 50micrometers, less than about 25 micrometers, less than about 10micrometers, or less than about 5 micrometers. The average diameter ofthe droplet may also be at least about 1 micrometer, at least about 2micrometers, at least about 3 micrometers, at least about 5 micrometers,at least about 10 micrometers, at least about 15 micrometers, or atleast about 20 micrometers in certain cases.

As used herein, a “fluid” is given its ordinary meaning, i.e., a liquidor a gas. Preferably a fluid is a liquid. The fluid may have anysuitable viscosity that permits flow. If two or more fluids are present,each fluid may be independently selected among essentially any fluids(liquids, gases, and the like) by those of ordinary skill in the art, byconsidering the relationship between the fluids. The fluids may each bemiscible or immiscible. For example, two fluids can be selected to beimmiscible within the time frame of formation of a stream of fluids, orwithin the time frame of reaction or interaction. Where the portionsremain liquid for a significant period of time then the fluids should besignificantly immiscible. Where, after contact and/or formation, thedispersed portions are quickly hardened by polymerization or the like,the fluids need not be as immiscible. Those of ordinary skill in the artcan select suitable miscible or immiscible fluids, using contact anglemeasurements or the like, to carry out the techniques of the invention.

As used herein, a first entity is “surrounded” by a second entity if aclosed loop can be drawn around the first entity through only the secondentity. A first entity is “completely surrounded” if closed loops goingthrough only the second entity can be drawn around the first entityregardless of direction. In one aspect, the first entity is a particle.In yet another aspect of the invention, the entities can both be fluids.For example, a hydrophilic liquid may be suspended in a hydrophobicliquid, a hydrophobic liquid may be suspended in a hydrophilic liquid, agas bubble may be suspended in a liquid, etc. Typically, a hydrophobicliquid and a hydrophilic liquid are substantially immiscible withrespect to each other, where the hydrophilic liquid has a greateraffinity to water than does the hydrophobic liquid. Examples ofhydrophilic liquids include, but are not limited to, water and otheraqueous solutions comprising water, such as cell or biological media,ethanol, salt solutions, etc. Examples of hydrophobic liquids include,but are not limited to, oils such as hydrocarbons, silicon oils,fluorocarbon oils, organic solvents etc.

The term “determining,” as used herein, generally refers to the analysisor measurement of a species, for example, quantitatively orqualitatively, or the detection of the presence or absence of thespecies. “Determining” may also refer to the analysis or measurement ofan interaction between two or more species, for example, quantitativelyor qualitatively, or by detecting the presence or absence of theinteraction. Example techniques include, but are not limited to,spectroscopy such as infrared, absorption, fluorescence, UV/visible,FTIR (“Fourier Transform Infrared Spectroscopy”), or Raman; gravimetrictechniques; ellipsometry; piezoelectric measurements; immunoassays;electrochemical measurements; optical measurements such as opticaldensity measurements; circular dichroism; light scattering measurementssuch as quasielectric light scattering; polarimetry; refractometry; orturbidity measurements.

General Techniques

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art (e.g., in cell culture, molecular genetics, nucleic acidchemistry, hybridisation techniques and biochemistry).

Standard techniques are used for molecular, genetic and biochemicalmethods (see generally, Sambrook et al., Molecular Cloning: A LaboratoryManual, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology(1999) 4^(th) Ed, John Wiley & Sons, Inc. which are incorporated hereinby reference) and chemical methods. In addition Harlow & Lane, ALaboratory Manual Cold Spring Harbor, N.Y., is referred to for standardImmunological Techniques.

(A) General Description

The microcapsules of the present invention require appropriate physicalproperties to allow the working of the invention.

First, to ensure that the compounds and the target may not diffusebetween microcapsules, the contents of each microcapsule must beisolated from the contents of the surrounding microcapsules, so thatthere is no or little exchange of compounds and target between themicrocapsules over the timescale of the experiment.

Second, the method of the present invention requires that there are onlya limited number of beads per microcapsule. This ensures that thecompounds and the target will be isolated from other beads.

Third, the formation and the composition of the microcapsules must notabolish the activity of the target.

Consequently, any microencapsulation system used must fulfill thesethree requirements. The appropriate system(s) may vary depending on theprecise nature of the requirements in each application of the invention,as will be apparent to the skilled person.

A wide variety of microencapsulation procedures are available (seeBenita, 1996) and may be used to create the microcapsules used inaccordance with the present invention. Indeed, more than 200microencapsulation methods have been identified in the literature(Finch, 1993).

These include membrane enveloped aqueous vesicles such as lipid vesicles(liposomes) (New, 1990) and non-ionic surfactant vesicles (van Hal etal., 1996). These are closed-membranous capsules of single or multiplebilayers of non-covalently assembled molecules, with each bilayerseparated from its neighbour by an aqueous compartment. In the case ofliposomes the membrane is composed of lipid molecules; these are usuallyphospholipids but sterols such as cholesterol may also be incorporatedinto the membranes (New, 1990). A variety of enzyme-catalysedbiochemical reactions, including RNA and DNA polymerisation, can beperformed within liposomes (Chakrabarti et al., 1994; Oberholzer et al.,1995a; Oberholzer et al., 1995b; Walde et al., 1994; Wick & Luisi,1996).

With a membrane-enveloped vesicle system much of the aqueous phase isoutside the vesicles and is therefore non-compartmentalised. Thiscontinuous, aqueous phase should be removed or the biological systems init inhibited or destroyed in order that the reactions are limited to themicrocapsules (Luisi et al., 1987).

Enzyme-catalysed biochemical reactions have also been demonstrated inmicrocapsules generated by a variety of other methods. Many enzymes areactive in reverse micellar solutions (Bru & Walde, 1991; Bru & Walde,1993; Creagh et al., 1993; Haber et al., 1993; Kumar et al., 1989; Luisi& B., 1987; Mao & Walde, 1991; Mao et al., 1992; Perez et al., 1992;Walde et al., 1994; Walde et al., 1993; Walde et al., 1988) such as theAOT-isooctane-water system (Menger & Yamada, 1979).

Microcapsules can also be generated by interfacial polymerisation andinterfacial complexation (Whateley, 1996). Microcapsules of this sortcan have rigid, nonpermeable membranes, or semipermeable membranes.Semipermeable microcapsules bordered by cellulose nitrate membranes,polyamide membranes and lipid-polyamide membranes can all supportbiochemical reactions, including multienzyme systems (Chang, 1987;Chang, 1992; Lim, 1984). Alginate/polylysine microcapsules (Lim & Sun,1980), which can be formed under very mild conditions, have also provento be very biocompatible, providing, for example, an effective method ofencapsulating living cells and tissues (Chang, 1992; Sun et al., 1992).

Non-membranous microencapsulation systems based on phase partitioning ofan aqueous environment in a colloidal system, such as an emulsion, mayalso be used.

Preferably, the microcapsules of the present invention are formed fromemulsions; heterogeneous systems of two immiscible liquid phases withone of the phases dispersed in the other as droplets of microscopic orcolloidal size (Becher, 1957; Sherman, 1968; Lissant, 1974; Lissant,1984).

Emulsions may be produced from any suitable combination of immiscibleliquids. Preferably the emulsion of the present invention has water(containing the biochemical components) as the phase present in the formof finely divided droplets (the disperse, internal or discontinuousphase) and a hydrophobic, immiscible liquid (an oil′) as the matrix inwhich these droplets are suspended (the nondisperse, continuous orexternal phase). Such emulsions are termed water-in-oil′ (W/O). This hasthe advantage that the entire aqueous phase containing the biochemicalcomponents is compartmentalised in discreet droplets (the internalphase). The external phase, being a hydrophobic oil, generally containsnone of the biochemical components and hence is inert.

The emulsion may be stabilised by addition of one or more surface-activeagents (surfactants). These surfactants are termed emulsifying agentsand act at the water/oil interface to prevent (or at least delay)separation of the phases. Many oils and many emulsifiers can be used forthe generation of water-in-oil emulsions; a recent compilation listedover 16,000 surfactants, many of which are used as emulsifying agents(Ash and Ash, 1993). Suitable oils include light white mineral oil anddecane. Suitable surfactants include: non-ionic surfactants (Schick,1966) such as sorbitan monooleate (Span.™.80; ICI), sorbitanmonostearate (Span.™.60; ICI), polyoxyethylenesorbitan monooleate(Tween.™. 80; ICI), and octylphenoxyethoxyethanol (Triton X-100); ionicsurfactants such as sodium cholate and sodium taurocholate and sodiumdeoxycholate; chemically inert silicone-based surfactants such aspolysiloxane-polycetyl-polyethylene glycol copolymer (Cetyl DimethiconeCopolyol) (e.g. Abil.™.EM90; Goldschmidt); and cholesterol.

Emulsions with a fluorocarbon (or perfluorocarbon) continuous phase(Krafft et al., 2003; Riess, 2002) may be particularly advantageous. Forexample, stable water-in-perfluorooctyl bromide andwater-in-perfluorooctylethane emulsions can be formed using F-alkyldimorpholinophosphates as surfactants (Sadtler et al., 1996).Non-fluorinated compounds are essentially insoluble in fluorocarbons andperfluorocarbons (Curran, 1998; Hildebrand and Cochran, 1949; Hudlicky,1992; Scott, 1948; Studer et al., 1997) and small drug-like molecules(typically <500 Da and Log P<5) (Lipinski et al., 2001) arecompartmentalised very effectively in the aqueous microcapsules ofwater-in-fluorocarbon and water-in-perfluorocarbon emulsions—with littleor no exchange between microcapsules.

Advantageously, compounds can be compartmentalised in microcapsulescomprising non-aqueous (organic) solvents. Non-fluorinated organicsolvents are essentially insoluble and immiscible with fluorocarbons andperfluorocarbons (Curran, 1998; Hildebrand and Cochran, 1949; Hudlicky,1992; Scott, 1948; Studer et al., 1997) allowing the formation ofemulsions with a fluorocarbon (or perfluorocarbon) continuous phase anda discontinous phase formed from a non-aqueous solvent such asdichloromethane, chloroform, carbon tetrachloride, toluene,tetrahydrofuran, diethyl ether, and ethanol. The ability to formsecondary compounds in microcapsules comprising non-aqueous solventsgreatly expands the repertoire of chemical reactions that can beperformed and secondary molecules that can be synthesised therein. Mostof synthetic organic chemistry is carried out in organic solventsincluding dichloromethane, chloroform, carbon tetrachloride, toluene,tetrahydrofuran, diethyl ether, and ethanol. Organic molecules dissolvebetter in organic solvents. Electrostatic interactions are enhanced inorganic solvents (due to the low dielectric constant), whereas they canbe solvated and made less reactive in aqueous solvents. For example,much of contemporary organic chemistry involves reactions relating tocarbonyl chemistry, including the use of metal enolates. Likewise for agrowing number of other organometallic interactions. These reactions areoften carried out under an inert atmosphere in anhydrous solvents(otherwise the reagents would be quenched by water). There are also alarge number of reactions which use palladium catalysis including theSuzuki reaction and the Heck reaction.

Creation of an emulsion generally requires the application of mechanicalenergy to force the phases together. There are a variety of ways ofdoing this which utilise a variety of mechanical devices, includingstirrers (such as magnetic stir-bars, propeller and turbine stirrers,paddle devices and whisks), homogenisers (including rotor-statorhomogenisers, high-pressure valve homogenisers and jet homogenisers),colloid mills, ultrasound and ‘membrane emulsification’ devices (Becher,1957; Dickinson, 1994).

Complicated biochemical processes, notably gene transcription andtranslation are also active in aqueous microcapsules formed inwater-in-oil emulsions. This has enabled compartmentalisation inwater-in-oil emulsions to be used for the selection of genes, which aretranscribed and translated in emulsion microcapsules and selected by thebinding or catalytic activities of the proteins they encode (Doi andYanagawa, 1999; Griffiths and Tawfik, 2003; Lee et al., 2002; Sepp etal., 2002; Tawfik and Griffiths, 1998). This was possible because theaqueous microcapsules formed in the emulsion were generally stable withlittle if any exchange of nucleic acids, proteins, or the products ofenzyme catalysed reactions between microcapsules.

The technology exists to create emulsions with volumes all the way up toindustrial scales of thousands of litres (Becher, 1957; Sherman, 1968;Lissant, 1974; Lissant, 1984).

The preferred microcapsule size will vary depending upon the preciserequirements of any individual screening process that is to be performedaccording to the present invention. In all cases, there will be anoptimal balance between the size of the compound library and thesensitivities of the assays to determine the identity of the compoundand target activity.

The size of emulsion microcapsules may be varied simply by tailoring theemulsion conditions used to form the emulsion according to requirementsof the screening system. The larger the microcapsule size, the larger isthe volume that will be required to encapsulate a given compoundlibrary, since the ultimately limiting factor will be the size of themicrocapsule and thus the number of microcapsules possible per unitvolume.

Water-in-oil emulsions can be re-emulsified to create water-in-oil-inwater double emulsions with an external (continuous) aqueous phase.These double emulsions can be analysed and, optionally, sorted using aflow cytometer (Bernath et al., 2004).

Highly monodisperse microcapsules can be produced using microfluidictechniques. For example, water-in-oil emulsions with less than 1.5%polydispersity can be generated by droplet break off in a co-flowingsteam of oil (Umbanhowar et al., 2000). Microfluidic systems can also beused for laminar-flow of aqueous microdroplets dispersed in a stream ofoil in microfluidic channels (Thorsen et al., 2001). This allows theconstruction of microfluidic devices for flow analysis and, optionally,flow sorting of microdroplets (Fu et al., 2002).

Advantageously, highly monodisperse microcapsules can be formed usingsystems and methods for the electronic control of fluidic species. [Oneaspect of the invention relates to systems and methods for producingdroplets of fluid surrounded by a liquid.

The fluid and the liquid may be essentially immiscible in many cases,i.e., immiscible on a time scale of interest (e.g., the time it takes afluidic droplet to be transported through a particular system ordevice). In certain cases, the droplets may each be substantially thesame shape or size, as further described below. The fluid may alsocontain other species, for example, certain molecular species (e.g., asfurther discussed below), cells, particles, etc.

In one set of embodiments, electric charge may be created on a fluidsurrounded by a liquid, which may cause the fluid to separate intoindividual droplets within the liquid. In some embodiments, the fluidand the liquid may be present in a channel, e.g., a microfluidicchannel, or other constricted space that facilitates application of anelectric field to the fluid (which may be “AC” or alternating current,“DC” or direct current etc.), for example, by limiting movement of thefluid with respect to the liquid. Thus, the fluid can be present as aseries of individual charged and/or electrically inducible dropletswithin the liquid. In one embodiment, the electric force exerted on thefluidic droplet may be large enough to cause the droplet to move withinthe liquid. In some cases, the electric force exerted on the fluidicdroplet may be used to direct a desired motion of the droplet within theliquid, for example, to or within a channel or a microfluidic channel(e.g., as further described herein), etc. As one example, in apparatus 5in FIG. 3A, droplets 15 created by fluid source 10 can be electricallycharged using an electric filed created by electric field generator 20.

Electric charge may be created in the fluid within the liquid using anysuitable technique, for example, by placing the fluid within an electricfield (which may be AC, DC, etc.), and/or causing a reaction to occurthat causes the fluid to have an electric charge, for example, achemical reaction, an ionic reaction, a photocatalyzed reaction, etc. Inone embodiment, the fluid is an electrical conductor. As used herein, a“conductor” is a material having a conductivity of at least about theconductivity of 18 megohm (MOhm or M.OMEGA.)) water. The liquidsurrounding the fluid may have a conductivity less than that of thefluid. For instance, the liquid may be an insulator, relative to thefluid, or at least a “leaky insulator,” i.e., the liquid is able to atleast partially electrically insulate the fluid for at least a shortperiod of time. Those of ordinary skill in the art will be able toidentify the conductivity of fluids. In one non-limiting embodiment, thefluid may be substantially hydrophilic, and the liquid surrounding thefluid may be substantially hydrophobic.

In some embodiments, the charge created on the fluid (for example, on aseries of fluidic droplets) may be at least about 10⁻²² C/micrometer³.In certain cases, the charge may be at least about 10⁻²¹ C/micrometer.³,and in other cases, the charge may be at least about 10⁻²⁰C/micrometer³, at least about 10⁻¹⁹ C/micrometer³, at least about 10⁻¹⁸C/micrometer³, at least about 10⁻¹⁷ C/micrometer³, at least about 10⁻¹⁶C/micrometer 3, at least about 10⁻¹⁵ C/micrometer3, at least about 10⁻¹⁴C/micrometer³, at least about 10⁻¹³ C/micrometer³, at least about 10⁻¹²C/micrometer³, at least about 10⁻¹¹ C/micrometer³, at least about 10⁻¹⁰C/micrometer³, or at least about 10⁻⁹ C/micrometer³ or more. In certainembodiments, the charge created on the fluid may be at least about 10⁻²¹C/micrometer², and in some cases, the charge may be at least about 10⁻²⁰C/micrometer², at least about 10⁻¹⁹ C/micrometer², at least about 10⁻¹⁸C/micrometer², at least about 10⁻¹⁷ C/micrometer², at least about 10⁻¹⁶C/micrometer², at least about 10⁻¹⁵ C/micrometer², at least about 10⁻¹⁴C/micrometer², or at least about 10⁻¹³ C/micrometer² or more. In otherembodiments, the charge may be at least about 10⁻¹⁴ C/droplet, and, insome cases, at least about 10⁻¹³ C/droplet, in other cases at leastabout 10⁻¹² C/droplet, in other cases at least about 10⁻¹¹ C/droplet, inother cases at least about 10⁻¹⁰ C/droplet, or in still other cases atleast about 10⁻⁹ C/droplet.

The electric field, in some embodiments, is generated from an electricfield generator, i.e., a device or system able to create an electricfield that can be applied to the fluid. The electric field generator mayproduce an AC field (i.e., one that varies periodically with respect totime, for example, sinusoidally, sawtooth, square, etc.), a DC field(i.e., one that is constant with respect to time), a pulsed field, etc.The electric field generator may be constructed and arranged to createan electric field within a fluid contained within a channel or amicrofluidic channel. The electric field generator may be integral to orseparate from the fluidic system containing the channel or microfluidicchannel, according to some embodiments. As used herein, “integral” meansthat portions of the components integral to each other are joined insuch a way that the components cannot be manually separated from eachother without cutting or breaking at least one of the components.

Techniques for producing a suitable electric field (which may be AC, DC,etc.) are known to those of ordinary skill in the art. For example, inone embodiment, an electric field is produced by applying voltage acrossa pair of electrodes, which may be positioned on or embedded within thefluidic system (for example, within a substrate defining the channel ormicrofluidic channel), and/or positioned proximate the fluid such thatat least a portion of the electric field interacts with the fluid. Theelectrodes can be fashioned from any suitable electrode material ormaterials known to those of ordinary skill in the art, including, butnot limited to, silver, gold, copper, carbon, platinum, copper,tungsten, tin, cadmium, nickel, indium tin oxide (“ITO”), etc., as wellas combinations thereof. In some cases, transparent or substantiallytransparent electrodes can be used. In certain embodiments, the electricfield generator can be constructed and arranged (e.g., positioned) tocreate an electric field applicable to the fluid of at least about 0.01V/micrometer, and, in some cases, at least about 0.03 V/micrometer, atleast about 0.05 V/micrometer, at least about 0.08 V/micrometer, atleast about 0.1 V/micrometer, at least about 0.3 V/micrometer, at leastabout 0.5 V/micrometer, at least about 0.7 V/micrometer, at least about1 V/micrometer, at least about 1.2 V/micrometer, at least about 1.4V/micrometer, at least about 1.6 V/micrometer, or at least about 2V/micrometer. In some embodiments, even higher electric fieldintensities may be used, for example, at least about 2 V/micrometer, atleast about 3 V/micrometer, at least about 5 V/micrometer, at leastabout 7 V/micrometer, or at least about 10 V/micrometer or more.

In some embodiments, an electric field may be applied to fluidicdroplets to cause the droplets to experience an electric force. Theelectric force exerted on the fluidic droplets may be, in some cases, atleast about 10⁻¹⁶ N/micrometer³. In certain cases, the electric forceexerted on the fluidic droplets may be greater, e.g., at least about10⁻¹⁵ N/micrometer³, at least about 10⁻¹⁴ N/micrometer³, at least about10⁻¹³ N/micrometer³, at least about 10⁻¹² N/micrometer³, at least about10⁻¹¹ N/micrometer³, at least about 10⁻¹⁰ N/micrometer³, at least about10⁻⁹ N/micrometer³, at least about 10⁻⁸ N/micrometer³, or at least about10⁻⁷ N/micrometer³ or more. In other embodiments, the electric forceexerted on the fluidic droplets, relative to the surface area of thefluid, may be at least about 10⁻¹⁵ N/micrometer², and in some cases, atleast about 10⁻¹⁴ N/micrometer², at least about 10⁻¹³ N/micrometer², atleast about 10⁻¹² N/micrometer², at least about 10⁻¹⁰ N/micrometer², atleast about 10-O N/micrometer², at least about 10⁻⁹ N/micrometer², atleast about 10⁻⁸ N/micrometer², at least about 10⁻⁷ N/micrometer², or atleast about 10⁻⁶ N/micrometer² or more. In yet other embodiments, theelectric force exerted on the fluidic droplets may be at least about10⁻⁹ N, at least about 10⁻⁸ N, at least about 10⁻⁷ N, at least about10⁻⁶ N, at least about 10⁻⁵ N, or at least about 10⁻⁴ N or more in somecases.

In some embodiments of the invention, systems and methods are providedfor at least partially neutralizing an electric charge present on afluidic droplet, for example, a fluidic droplet having an electriccharge, as described above. For example, to at least partiallyneutralize the electric charge, the fluidic droplet may be passedthrough an electric field and/or brought near an electrode, e.g., usingtechniques such as those described herein. Upon exiting of the fluidicdroplet from the electric field (i.e., such that the electric field nolonger has a strength able to substantially affect the fluidic droplet),and/or other elimination of the electric field, the fluidic droplet maybecome electrically neutralized, and/or have a reduced electric charge.

In another set of embodiments, droplets of fluid can be created from afluid surrounded by a liquid within a channel by altering the channeldimensions in a manner that is able to induce the fluid to formindividual droplets. The channel may, for example, be a channel thatexpands relative to the direction of flow, e.g., such that the fluiddoes not adhere to the channel walls and forms individual dropletsinstead, or a channel that narrows relative to the direction of flow,e.g., such that the fluid is forced to coalesce into individualdroplets. One example is shown in FIG. 7A, where channel 510 includes aflowing fluid 500 (flowing downwards), surrounded by liquid 505. Channel510 narrows at location 501, causing fluid 500 to form a series ofindividual fluidic droplets 515. In other embodiments, internalobstructions may also be used to cause droplet formation to occur. Forinstance, baffles, ridges, posts, or the like may be used to disruptliquid flow in a manner that causes the fluid to coalesce into fluidicdroplets.

In some cases, the channel dimensions may be altered with respect totime (for example, mechanically or electromechanically, pneumatically,etc.) in such a manner as to cause the formation of individual fluidicdroplets to occur. For example, the channel may be mechanicallycontracted (“squeezed”) to cause droplet formation, or a fluid streammay be mechanically disrupted to cause droplet formation, for example,through the use of moving baffles, rotating blades, or the like. As anon-limiting example, in FIG. 7B, fluid 500 flows through channel 510 ina downward direction. Fluid 500 is surrounded by liquid 505.Piezoelectric devices 520 positioned near or integral to channel 510 maythen mechanically constrict or “squeeze” channel 510, causing fluid 500to break up into individual fluidic droplets 515.

In yet another set of embodiments, individual fluidic droplets can becreated and maintained in a system comprising three essentially mutuallyimmiscible fluids (i.e., immiscible on a time scale of interest), whereone fluid is a liquid carrier, and the second fluid and the third fluidalternate as individual fluidic droplets within the liquid carrier. Insuch a system, surfactants are not necessarily required to ensureseparation of the fluidic droplets of the second and third fluids. As anexample, with reference to FIG. 14A, within channel 700, a first fluid701 and a second fluid 702 are each carried within liquid carrier 705.First fluid 701 and second fluid 702 alternate as a series ofalternating, individual droplets, each carried by liquid carrier 705within channel 700. As the first fluid, the second fluid, and the liquidcarrier are all essentially mutually immiscible, any two of the fluids(or all three fluids) can come into contact without causing dropletcoalescence to occur. A photomicrograph of an example of such a systemis shown in FIG. 14B, illustrating first fluid 701 and second fluid 702,present as individual, alternating droplets, each contained withinliquid carrier 705.

One example of a system involving three essentially mutually immisciblefluids is a silicone oil, a mineral oil, and an aqueous solution (i.e.,water, or water containing one or more other species that are dissolvedand/or suspended therein, for example, a salt solution, a salinesolution, a suspension of water containing particles or cells, or thelike). Another example of a system is a silicone oil, a fluorocarbonoil, and an aqueous solution. Yet another example of a system is ahydrocarbon oil (e.g., hexadecane), a fluorocarbon oil, and an aqueoussolution. In these examples, any of these fluids may be used as theliquid carrier. Non-limiting examples of suitable fluorocarbon oilsinclude octadecafluorodecahydronaphthalene:

A non-limiting example of such a system is illustrated in FIG. 14B. Inthis figure, fluidic network 710 includes a channel containing liquidcarrier 705, and first fluid 701 and second fluid 702. Liquid carrier705 is introduced into fluidic network 710 through inlet 725, whilefirst fluid 701 is introduced through inlet 721, and second fluid 702 isintroduced through inlet 722. Channel 716 within fluidic network 710contains liquid carrier 715 introduced from inlet 725. Initially, firstfluid 701 is introduced into liquid carrier 705, forming fluidicdroplets therein. Next, second fluid 702 is introduced into liquid 705,forming fluidic droplets therein that are interspersed with the fluidicdroplets containing first fluid 701. Thus, upon reaching channel 717,liquid carrier 705 contains a first set of fluidic droplets containingfirst fluid 701, interspersed with a second set of fluidic dropletscontaining second fluid 702. In the embodiment illustrated, channel 706optionally comprises a series of bends, which may allow mixing to occurwithin each of the fluidic droplets, as further discussed below.However, it should be noted that in this embodiment, since first fluid701 and second fluid 702 are essentially immiscible, significant fusionand/or mixing of the droplets containing first fluid 701 with thedroplets containing second fluid 702 is not generally expected.

Other examples of the production of droplets of fluid surrounded by aliquid are described in International Patent Application Serial No.PCT/US2004/010903, filed Apr. 9, 2004 by Link, et al. and InternationalPatent Application Serial No. PCT/US03/20542, filed Jun. 30, 2003 byStone, et al., published as WO 2004/002627 on Jan. 8, 2004, eachincorporated herein by reference.

In some embodiments, the fluidic droplets may each be substantially thesame shape and/or size. The shape and/or size can be determined, forexample, by measuring the average diameter or other characteristicdimension of the droplets. The term “determining,” as used herein,generally refers to the analysis or measurement of a species, forexample, quantitatively or qualitatively, and/or the detection of thepresence or absence of the species. “Determining” may also refer to theanalysis or measurement of an interaction between two or more species,for example, quantitatively or qualitatively, or by detecting thepresence or absence of the interaction. Examples of suitable techniquesinclude, but are not limited to, spectroscopy such as infrared,absorption, fluorescence, UV/visible, FTIR (“Fourier Transform InfraredSpectroscopy”), or Raman; gravimetric techniques; ellipsometry;piezoelectric measurements; immunoassays; electrochemical measurements;optical measurements such as optical density measurements; circulardichroism; light scattering measurements such as quasielectric lightscattering; polarimetry; refractometry; or turbidity measurements.

The “average diameter” of a plurality or series of droplets is thearithmetic average of the average diameters of each of the droplets.Those of ordinary skill in the art will be able to determine the averagediameter (or other characteristic dimension) of a plurality or series ofdroplets, for example, using laser light scattering, microscopicexamination, or other known techniques. The diameter of a droplet, in anon-spherical droplet, is the mathematically-defined average diameter ofthe droplet, integrated across the entire surface. The average diameterof a droplet (and/or of a plurality or series of droplets) may be, forexample, less than about 1 mm, less than about 500 micrometers, lessthan about 200 micrometers, less than about 100 micrometers, less thanabout 75 micrometers, less than about 50 micrometers, less than about 25micrometers, less than about 10 micrometers, or less than about 5micrometers in some cases. The average diameter may also be at leastabout 1 micrometer, at least about 2 micrometers, at least about 3micrometers, at least about 5 micrometers, at least about 10micrometers, at least about 15 micrometers, or at least about 20micrometers in certain cases.

In certain instances, the invention provides for the production ofdroplets consisting essentially of a substantially uniform number ofentities of a species therein (i.e., molecules, compounds, cells,particles, etc.). For example, about 90%, about 93%, about 95%, about97%, about 98%, or about 99%, or more of a plurality or series ofdroplets may each contain the same number of entities of a particularspecies. For instance, a substantial number of fluidic dropletsproduced, e.g., as described above, may each contain 1 entity, 2entities, 3 entities, 4 entities, 5 entities, 7 entities, 10 entities,15 entities, 20 entities, 25 entities, 30 entities, 40 entities, 50entities, 60 entities, 70 entities, 80 entities, 90 entities, 100entities, etc., where the entities are molecules or macromolecules,cells, particles, etc. In some cases, the droplets may eachindependently contain a range of entities, for example, less than 20entities, less than 15 entities, less than 10 entities, less than 7entities, less than 5 entities, or less than 3 entities in some cases.In one set of embodiments, in a liquid containing droplets of fluid,some of which contain a species of interest and some of which do notcontain the species of interest, the droplets of fluid may be screenedor sorted for those droplets of fluid containing the species as furtherdescribed below (e.g., using fluorescence or other techniques such asthose described above), and in some cases, the droplets may be screenedor sorted for those droplets of fluid containing a particular number orrange of entities of the species of interest, e.g., as previouslydescribed. Thus, in some cases, a plurality or series of fluidicdroplets, some of which contain the species and some of which do not,may be enriched (or depleted) in the ratio of droplets that do containthe species, for example, by a factor of at least about 2, at leastabout 3, at least about 5, at least about 10, at least about 15, atleast about 20, at least about 50, at least about 100, at least about125, at least about 150, at least about 200, at least about 250, atleast about 500, at least about 750, at least about 1000, at least about2000, or at least about 5000 or more in some cases. In other cases, theenrichment (or depletion) may be in a ratio of at least about 10⁴, atleast about 10⁵, at least about 10⁶, at least about 10⁷, at least about10⁸, at least about 10⁹, at least about 10¹⁰, at least about 10¹¹, atleast about 10¹², at least about 10¹³, at least about 10¹⁴, at leastabout 10¹⁵, or more. For example, a fluidic droplet containing aparticular species may be selected from a library of fluidic dropletscontaining various species, where the library may have about 10⁵, about10⁶, about 10⁷, about 10⁸, about 10⁹, about 10¹⁰, about 10¹¹, about10¹², about 10¹³, about 10¹⁴, about 10¹⁵, or more items, for example, aDNA library, an RNA library, a protein library, a combinatorialchemistry library, etc. In certain embodiments, the droplets carryingthe species may then be fused, reacted, or otherwise used or processed,etc., as further described below, for example, to initiate or determinea reaction.

The use of microfluidic handling to create microcapsoules according tothe invention has a number of advantages:

-   -   a) They allow the formation of highly monodisperse microcapsules        (<1.5% polydispersity), each of which functions as an almost        identical, very small microreactor,    -   b) The microcapsules can have volumes ranging from about 1        femtolitre to about 1 nanolitre.    -   c) Compartmentalisation in microcapsules prevents diffusion and        dispersion due to parabolic flow.    -   d) By using a perfluorocarbon carrier fluid it is possible to        prevent exchange of molecules between microcapsules.    -   e) Compounds in microcapsules cannot react or interact with the        fabric of the microchannels as they are separated by a layer of        inert perfluorocarbon carrier fluid.    -   f) Microcapsules can be created at up to and including 10,000        s⁻¹ and screened using optical methods at the same rate. This is        a throughput of ˜10⁹ per day.

Microcapsules can, advantageously, be fused or split. For example,aqueous microdroplets can be merged and split using microfluidicssystems (Link et al., 2004; Song et al., 2003). Microcapsule fusionallows the mixing of reagents. Fusion, for example, of a microcapsulecontaining the target with a microcapsule containing the compound couldinitiate the reaction between target and compound. Microcapsulesplitting allows single microcapsules to be split into two or moresmaller microcapsules. For example a single microcapsule containing acompound can be split into multiple microcapsules which can then each befused with a different microcapsule containing a different target. Asingle microcapsule containing a target can also be split into multiplemicrocapsules which can then each be fused with a different microcapsulecontaining a different compound, or compounds at differentconcentrations.

In one aspect, the invention relates to microfluidic systems and methodsfor splitting a fluidic droplet into two or more droplets. The fluidicdroplet may be surrounded by a liquid, e.g., as previously described,and the fluid and the liquid are essentially immiscible in some cases.The two or more droplets created by splitting the original fluidicdroplet may each be substantially the same shape and/or size, or the twoor more droplets may have different shapes and/or sizes, depending onthe conditions used to split the original fluidic droplet. In manycases, the conditions used to split the original fluidic droplet can becontrolled in some fashion, for example, manually or automatically(e.g., with a processor, as discussed below). In some cases, eachdroplet in a plurality or series of fluidic droplets may beindependently controlled. For example, some droplets may be split intoequal parts or unequal parts, while other droplets are not split.

According to one set of embodiments, a fluidic droplet can be splitusing an applied electric field. The electric field may be an AC field,a DC field, etc. The fluidic droplet, in this embodiment, may have agreater electrical conductivity than the surrounding liquid, and, insome cases, the fluidic droplet may be neutrally charged. In someembodiments, the droplets produced from the original fluidic droplet areof approximately equal shape and/or size. In certain embodiments, in anapplied electric field, electric charge may be urged to migrate from theinterior of the fluidic droplet to the surface to be distributedthereon, which may thereby cancel the electric field experienced in theinterior of the droplet. In some embodiments, the electric charge on thesurface of the fluidic droplet may also experience a force due to theapplied electric field, which causes charges having opposite polaritiesto migrate in opposite directions.

The charge migration may, in some cases, cause the drop to be pulledapart into two separate fluidic droplets. The electric field applied tothe fluidic droplets may be created, for example, using the techniquesdescribed above, such as with a reaction an electric field generator,etc.

As a non-limiting example, in FIG. 1A, where no electric field isapplied, fluidic droplets 215 contained in channel 230 are carried by asurrounding liquid, which flows towards intersection 240, leading tochannels 250 and 255. In this example, the surrounding liquid flowsthrough channels 250 and 255 at equal flowrates. Thus, at intersection240, fluidic droplets 215 do not have a preferred orientation ordirection, and move into exit channels 250 and 255 with equalprobability due to the surrounding liquid flow. In contrast, 1 in FIG.1B, while the surrounding liquid flows in the same fashion as FIG. 1A,under the influence of an applied electric field of 1.4 V/micrometers,fluidic droplets 215 are split into two droplets at intersection 240,forming new droplets 216 and 217. Droplet 216 moves to the left inchannel 250, while droplet 217 moves to the right in channel 255.

A schematic of this process can be seen in FIG. 5, where a neutralfluidic droplet 530, surrounded by a liquid 535 in channel 540, issubjected to applied electric field 525, created by electrodes 526 and527. Electrode 526 is positioned near channel 542, while electrode 527is positioned near channel 544. Under the influence of electric field525, charge separation is induced within fluidic droplet 530, i.e., suchthat a positive charge is induced at one end of the droplet, while anegative charge is induced at the other end of the droplet. The dropletmay then split into a negatively charged droplet 545 and a positivelycharged droplet 546, which then may travel in channels 542 and 544,respectively. In some cases, one or both of the electric charges on theresulting charged droplets may also be neutralized, as previouslydescribed.

Other examples of splitting a fluidic droplet into two droplets aredescribed in International Patent Application Serial No.PCT/US2004/010903, filed Apr. 9, 2004 by Link, et al.; U.S. ProvisionalPatent Application Ser. No. 60/498,091, filed Aug. 27, 2003, by Link,et. al.; and International Patent Application Serial No. PCT/US03/20542,filed Jun. 30, 2003 by Stone, et al., published as WO 2004/002627 onJan. 8, 2004, each incorporated herein by reference.

The invention, in yet another aspect, relates to systems and methods forfusing or coalescing two or more fluidic droplets into one droplet. Forexample, in one set of embodiments, systems and methods are providedthat are able to cause two or more droplets (e.g., arising fromdiscontinuous streams of fluid) to fuse or coalesce into one droplet incases where the two or more droplets ordinarily are unable to fuse orcoalesce, for example, due to composition, surface tension, dropletsize, the presence or absence of surfactants, etc. In certainmicrofluidic systems, the surface tension of the droplets, relative tothe size of the droplets, may also prevent fusion or coalescence of thedroplets from occurring in some, cases.

In one embodiment, two fluidic droplets may be given opposite electriccharges (i.e., positive and negative charges, not necessarily of thesame magnitude), which may increase the electrical interaction of thetwo droplets such that fusion or coalescence of the droplets can occurdue to their opposite electric charges, e.g., using the techniquesdescribed herein. For instance, an electric field may be applied to thedroplets, the droplets may be passed through a capacitor, a chemicalreaction may cause the droplets to become charged, etc. As an example,as is shown schematically in FIG. 13A, uncharged droplets 651 and 652,carried by a liquid 654 contained within a microfluidic channel 653, arebrought into contact with each other, but the droplets are not able tofuse or coalesce, for instance, due to their size and/or surfacetension. The droplets, in some cases, may not be able to fuse even if asurfactant is applied to lower the surface tension of the droplets.However, if the fluidic droplets are electrically charged with oppositecharges (which can be, but are not necessarily of, the same magnitude),the droplets may be able to fuse or coalesce. For instance, in FIG. 13B,positively charged droplets 655 and negatively charged droplets 656 aredirected generally towards each other such that the electricalinteraction of the oppositely charged droplets causes the droplets tofuse into fused droplets 657.

In another embodiment, the fluidic droplets may not necessarily be givenopposite electric charges (and, in some cases, may not be given anyelectric charge), and are fused through the use of dipoles induced inthe fluidic droplets that causes the fluidic droplets to coalesce. Inthe example illustrated in FIG. 13C, droplets 660 and 661 (which mayeach independently be electrically charged or neutral), surrounded byliquid 665 in channel 670, move through the channel such that they arethe affected by an applied electric field 675. Electric field 675 may bean AC field, a DC field, etc., and may be created, for instance, usingelectrodes 676 and 677, as shown here. The induced dipoles in each ofthe fluidic droplets, as shown in FIG. 13C, may cause the fluidicdroplets to become electrically attracted towards each other due totheir local opposite charges, thus causing droplets 660 and 661 to fuseto produce droplet 663. In FIG. 13D, droplets 651 and 652 flow togetherto fuse to form droplet 653, which flows in a third channel.

It should be noted that, in various embodiments, the two or moredroplets allowed to coalesce are not necessarily required to meet“head-on.” Any angle of contact, so long as at least some fusion of thedroplets initially occurs, is sufficient. As an example, in FIG. 12H,droplets 73 and 74 each are traveling in substantially the samedirection (e.g., at different velocities), and are able to meet andfuse. As another example, in FIG. 121, droplets 73 and 74 meet at anangle and fuse. In FIG. 12J, three fluidic droplets 73, 74 and 68 meetand fuse to produce droplet 79.

Other examples of fusing or coalescing fluidic droplets are described inInternational Patent Application Serial No. PCT/JS2004/010903, filedApr. 9, 2004 by Link, et al., incorporated herein by reference.

Fluidic handling of microcapsules therefore results in furtheradvantages:

-   -   a) Microcapsules can be split into two or more smaller        microdroplets allowing the reagents contained therein to be        reacted with a series of different molecules in parallel or        assayed in multiplicate.    -   b) Microcapsules can be fused. This allows molecules to be: (a)        diluted, (b) mixed with other molecules, and (c) reactions        initiated, terminated or modulated at precisely defined times.    -   c) Reagents can be mixed very rapidly (in <2 ms) in        microcapsules using chaotic advection, allowing fast kinetic        measurements and very high throughput.    -   d) Reagents can be mixed in a combinatorial manner. For example,        allowing the effect of all possible pairwise combinations of        compounds in a compound library on a target to be tested

Creating and manipulated microcapsules in microfluidic systems meansthat:

-   -   a) Stable streams of microcapsules can be formed in        microchannels and identified by their relative positions.    -   b) If the reactions are accompanied by an optical signal (e.g. a        change in fluorescence) a spatially-resolved optical image of        the microfluidic network allows time resolved measurements of        the reactions in each microcapsules.    -   c) Microcapsules can be separated using a microfluidic flow        sorter to allow recovery and further analysis or manipulation of        the molecules they contain.        Screening/Sorting of Microcapsules

In still another aspect, the invention provides systems and methods forscreening or sorting fluidic droplets in a liquid, and in some cases, atrelatively high rates. For example, a characteristic of a droplet may besensed and/or determined in some fashion (e.g., as further describedbelow), then the droplet may be directed towards a particular region ofthe device, for example, for sorting or screening purposes.

In some embodiments, a characteristic of a fluidic droplet may be sensedand/or determined in some fashion, for example, as described herein(e.g., fluorescence of the fluidic droplet may be determined), and, inresponse, an electric field may be applied or removed from the fluidicdroplet to direct the fluidic droplet to a particular region (e.g. achannel). In some cases, high sorting speeds may be achievable usingcertain systems and methods of the invention. For instance, at leastabout 10 droplets per second may be determined and/or sorted in somecases, and in other cases, at least about 20 droplets per second, atleast about 30 droplets per second, at least about 100 droplets persecond, at least about 200 droplets per second, at least about 300droplets per second, at least about 500 droplets per second, at leastabout 750 droplets per second, at least about 1000 droplets per second,at least about 1500 droplets per second, at least about 2000 dropletsper second, at least about 3000 droplets per second, at least about 5000droplets per second, at least about 7500 droplets per second, at leastabout 10,000 droplets per second, at least about 15,000 droplets persecond, at least about 20,000 droplets per second, at least about 30,000droplets per second, at least about 50,000 droplets per second, at leastabout 75,000 droplets per second, at least about 100,000 droplets persecond, at least about 150,000 droplets per second, at least about200,000 droplets per second, at least about 300,000 droplets per second,at least about 500,000 droplets per second, at least about 750,000droplets per second, at least about 1,000,000 droplets per second, atleast about 1,500,000 droplets per second, at least about 2,000,000 ormore droplets per second, or at least about 3,000,000 or more dropletsper second may be determined and/or sorted in such a fashion.

In one set of embodiments, a fluidic droplet may be directed by creatingan electric charge (e.g., as previously described) on the droplet, andsteering the droplet using an applied electric field, which may be an ACfield, a DC field, etc. As an example, in reference to FIGS. 2-4, anelectric field may be selectively applied and removed (or a differentelectric field may be applied, e.g., a reversed electric field as shownin FIG. 4A) as needed to direct the fluidic droplet to a particularregion. The electric field may be selectively applied and removed asneeded, in some embodiments, without substantially altering the flow ofthe liquid containing the fluidic droplet. For example, a liquid mayflow on a substantially steady-state basis (i.e., the average flowrateof the liquid containing the fluidic droplet deviates by less than 20%or less than 15% of the steady-state flow or the expected value of theflow of liquid with respect to time, and in some cases, the averageflowrate may deviate less than 10% or less than 5%) or otherpredetermined basis through a fluidic system of the invention (e.g.,through a channel or a microchannel), and fluidic droplets containedwithin the liquid may be directed to various regions, e.g., using anelectric field, without substantially altering the flow of the liquidthrough the fluidic system. As a particular example, in FIGS. 2A, 3A and4A, a liquid containing fluidic droplets 15 flows from fluid source 10,through channel 30 to intersection 40, and exits through channels 50 and55. In FIG. 2A, fluidic droplets 15 are directed through both channels50 and 55, while in FIG. 3A, fluidic droplets 15 are directed to onlychannel 55 and, in FIG. 4A, fluidic droplets 15 are directed to onlychannel 50.

In another set of embodiments, a fluidic droplet may be sorted orsteered by inducing a dipole in the fluidic droplet (which may beinitially charged or uncharged), and sorting or steering the dropletusing an applied electric field. The electric field may be an AC field,a DC field, etc. For example, with reference to FIG. 9A, a channel 540,containing fluidic droplet 530 and liquid 535, divides into channel 542and 544. Fluidic droplet 530 may have an electric charge, or it may beuncharged. Electrode 526 is positioned near channel 542, while electrode527 is positioned near channel 544. Electrode 528 is positioned near thejunction of channels 540, 542, and 544. In FIGS. 9C and 9D, a dipole isinduced in the fluidic droplet using electrodes 526, 527, and/or 528. InFIG. 9C, a dipole is induced in droplet 530 by applying an electricfield 525 to the droplet using electrodes 527 and 528. Due to thestrength of the electric field, the droplet is strongly attracted to theright, into channel 544. Similarly, in FIG. 9D, a dipole is induced indroplet 530 by applying an electric field 525 to the droplet usingelectrodes 526 and 528, causing the droplet to be attracted into channel542. Thus, by applying the proper electric field, droplet 530 can bedirected to either channel 542 or 544 as desired.

In other embodiments, however, the fluidic droplets may be screened orsorted within a fluidic system of the invention by altering the flow ofthe liquid containing the droplets. For instance, in one set ofembodiments, a fluidic droplet may be steered or sorted by directing theliquid surrounding the fluidic droplet into a first channel, a secondchannel, etc. As a non-limiting example, with reference to FIG. 10A,fluidic droplet 570 is surrounded by a liquid 575 in channel 580.Channel 580 divides into three channels 581, 582, and 583. The flow ofliquid 575 can be directed into any of channels 581, 582, and 583 asdesired, for example, using flow-controlling devices known to those ofordinary skill in the art, for example, valves, pumps, pistons, etc.Thus, in FIG. 10B, fluidic droplet 570 is directed into channel 581 bydirecting liquid 575 to flow into channel 581 (indicated by arrows 574);in FIG. 10C, fluidic droplet 570 is directed into channel 582 bydirecting liquid 575 to flow into channel 582 (indicated by arrows 574);and in FIG. 10D, fluidic droplet 570 is directed into channel 583 bydirecting liquid 575 to flow into channel 583 (indicated by arrows 574).

However, it is preferred that control of the flow of liquids inmicrofluidic systems is not used to direct the flow of fluidic dropletstherein, but that an alternative method is used. Advantageously,therefore, the microcapsules are not sorted by altering the direction ofthe flow of a carrier fluid in a microfluidic system.

In another set of embodiments, pressure within a fluidic system, forexample, within different channels or within different portions of achannel, can be controlled to direct the flow of fluidic droplets. Forexample, a droplet can be directed toward a channel junction includingmultiple options for further direction of flow (e.g., directed toward abranch, or fork, in a channel defining optional downstream flowchannels). Pressure within one or more of the optional downstream flowchannels can be controlled to direct the droplet selectively into one ofthe channels, and changes in pressure can be effected on the order ofthe time required for successive droplets to reach the junction, suchthat the downstream flow path of each successive droplet can beindependently controlled. In one arrangement, the expansion and/orcontraction of liquid reservoirs may be used to steer or sort a fluidicdroplet into a channel, e.g., by causing directed movement of the liquidcontaining the fluidic droplet. The liquid reservoirs may be positionedsuch that, when activated, the movement of liquid caused by theactivated reservoirs causes the liquid to flow in a preferred direction,carrying the fluidic droplet in that preferred direction. For instance,the expansion of a liquid reservoir may cause a flow of liquid towardsthe reservoir, while the contraction of a liquid reservoir may cause aflow of liquid away from the reservoir. In some cases, the expansionand/or contraction of the liquid reservoir may be combined with otherflow-controlling devices and methods, e.g., as described herein.Non-limiting examples of devices able to cause the expansion and/orcontraction of a liquid reservoir include pistons and piezoelectriccomponents. In some cases, piezoelectric components may be particularlyuseful due to their relatively rapid response times, e.g., in responseto an electrical signal.

As a non-limiting example, in FIG. 11A, fluidic droplet 600 issurrounded by a liquid 605 in channel 610. Channel 610 divides intochannels 611, 612. Positioned in fluidic communication with channels 611and 612 are liquid reservoirs 617 and 618, which may be expanded and/orcontracted, for instance, by piezoelectric components 615 and 616, by apiston (not shown), etc. In FIG. 1B, liquid reservoir 617 has beenexpanded, while liquid reservoir 618 has been contracted. The effect ofthe expansion/contractions of the reservoirs is to cause a net flow ofliquid towards channel 611, as indicated by arrows 603. Thus, fluidicdroplet 600, upon reaching the junction between the channels, isdirected to channel 611 by the movement of liquid 605. The reversesituation is shown in FIG. 11C, where liquid reservoir 617 hascontracted while liquid reservoir 618 has been expanded. A net flow ofliquid occurs towards channel 612 (indicated by arrows 603), causingfluidic droplet 600 to move into channel 612. It should be noted,however, that reservoirs 617 and 618 do not both need to be activated todirect fluidic droplet 600 into channels 611 or 612. For example, in oneembodiment, fluidic droplet 600 may be directed to channel 611 by theexpansion of liquid reservoir 617 (without any alteration of reservoir618), while in another embodiment, fluidic droplet 600 may be directedto channel 611 by the contraction of liquid reservoir 618 (without anyalteration of reservoir 617). In some cases, more than two liquidreservoirs may be used.

In some embodiments, the fluidic droplets may be sorted into more thantwo channels. Non-limiting examples of embodiments of the inventionhaving multiple regions within a fluidic system for the delivery ofdroplets are shown in FIGS. 6A and 6B. Other arrangements are shown inFIGS. 10A-10D. In FIG. 6A, charged droplets 315 in channel 330 may bedirected as desired to any one of exit channels 350, 352, 354, or 356,by applying electric fields to control the movement of the droplets atintersections 340, 341, and 342, using electrodes 321/322, 323/324, and325/326, respectively. In FIG. 6A, droplets 315 are directed to channel354 using applied electric fields 300 and 301, using principles similarto those discussed above. Similarly, in FIG. 6B, charged droplets 415 inchannel 430 can be directed to any one of exit channels 450, 452, 454,456, or 458, by applying electric fields to control the movement of thedroplets at intersections 440, 441, 442, and 443, using electrodes421/422, 423/424, 425/426, and 427/428, respectively. In this figure,droplets 415 are directed to channel 454; of course, the chargeddroplets may be directed to any other exit channel as desired.

In another example, in apparatus 5, as schematically illustrated in FIG.2A, fluidic droplets 15 created by fluid source 10 are positivelycharged due to an applied electric field created using electric fieldgenerator 20, which comprises two electrodes 22, 24.

Fluidic droplets 15 are directed through channel 30 by a liquidcontaining the droplets, and are directed towards intersection 40. Atintersection 40, the fluidic droplets do not have a preferredorientation or direction, and move into exit channels 50 and 55 withequal probability (in this embodiment, liquid drains through both exitchannels 50 and 55 at substantially equal rates). Similarly, fluidicdroplets 115 created by fluid source 110 are negatively charged due toan applied electric field created using electric field generator 120,which comprises electrodes 122 and 124. After traveling through channel130 towards intersection 140, the fluidic droplets do not have apreferred orientation or direction, and move into exit channels 150 and155 with equal probability, as the liquid exits through exit channels150 and 155 at substantially equal rates. A representativephotomicrograph of intersection 140 is shown in FIG. 2B.

In the schematic diagram of FIG. 3A, an electric field 100 of 1.4V/micrometer has been applied to apparatus 5 of FIG. 2A, in a directiontowards the right of apparatus 5. Positively-charged fluidic droplets 15in channel 30, upon reaching intersection 40, are directed to the rightin channel 55 due to the applied electric field 100, while the liquidcontaining the droplets continues to exit through exit channels 50 and55 at substantially equal rates. Similarly, negatively-charged fluidicdroplets 115 in channel 130, upon reaching intersection 140, aredirected to the left in channel 150 due to the applied electric field100, while the liquid fluid continues to exit the device through exitchannels 150 and 155 at substantially equal rates. Thus, electric field100 can be used to direct fluidic droplets into particular channels asdesired. A representative photomicrograph of intersection 140 is shownin FIG. 3B.

FIG. 4A is a schematic diagram of apparatus 5 of FIG. 2A, also with anapplied electric field 100 of 1.4 V/micrometer, but in the oppositedirection (i.e., −1.4 V/micrometer). In this figure, positively-chargedfluidic droplets 15 in channel 30, upon reaching intersection 40, aredirected to the left into channel 50 due to the applied electric field100, while negatively-charged fluidic droplets 115 in channel 130, uponreaching intersection 140, are directed to the right into channel 155due to applied electric field 100. The liquid containing the dropletsexits through exit channels 50 and 55, and 150 and 155, at substantiallyequal rates. A representative photomicrograph of intersection 140 isshown in FIG. 4B.

In some embodiments of the invention, a fluidic droplet may be sortedand/or split into two or more separate droplets, for example, dependingon the particular application. Any of the above-described techniques maybe used to spilt and/or sort droplets. As a non-limiting example, byapplying (or removing) a first electric field to a device (or a portionthereof), a fluidic droplet may be directed to a first region orchannel; by applying (or removing) a second electric field to the device(or a portion thereof), the droplet may be directed to a second regionor channel; by applying a third electric field to the device (or aportion thereof), the droplet may be directed to a third region orchannel; etc., where the electric fields may differ in some way, forexample, in intensity, direction, frequency, duration, etc. In a seriesof droplets, each droplet may be independently sorted and/or split; forexample, some droplets may be directed to one location or another, whileother droplets may be split into multiple droplets directed to two ormore locations.

As one particular example, in FIG. 8A, fluidic droplet 550, surroundingliquid 555 in channel 560 may be directed to channel 556, channel 557,or be split in some fashion between channels 562 and 564. In FIG. 8B, bydirecting surrounding liquid 555 towards channel 562, fluidic droplet550 may be directed towards the left into channel 562; in FIG. 8C, bydirecting surrounding liquid 555 towards channel 564, fluidic droplet550 may be directed towards the right into channel 564. In FIG. 8D, anelectric field may be applied, in combination with control of the flowof liquid 555 surrounding fluidic droplet 550, that causes the dropletto impactjunction 561, which may cause the droplet to split into twoseparate fluidic droplets 565, 566. Fluidic droplet 565 is directed tochannel 562, while fluidic droplet 566 is directed to channel 564. Ahigh degree of control of the applied electric field may be achieved tocontrol droplet formation; thus, for example, after fluidic droplet 565has been split into droplets 565 and 566, droplets 565 and 566 may be ofsubstantially equal size, or either of droplets 565 and 566 may belarger, e.g., as is shown in FIGS. 8E and 8F, respectively.

As another example, in FIG. 9A, channel 540, carrying fluidic droplet530 and liquid 535, divides into channel 542 and 544. Fluidic droplet530 may be electrically charged, or it may uncharged. Electrode 526 ispositioned near channel 542, while electrode 527 is positioned nearchannel 544. Electrode 528 is positioned near the junction of channels540, 542, and 544. When fluidic droplet 530 reaches the junction, it maybe subjected to an electric field, and/or directed to a channel or otherregion, for example, by directing the surrounding liquid into thechannel. As shown in FIG. 9B, fluidic droplet 530 may be split into twoseparate droplets 565 and 566 by applying an electric field 525 to thedroplet using electrodes 526 and 527. In FIG. 9C, a dipole can beinduced in droplet 530 by applying an electric field 525 to the dropletusing electrodes 527 and 528. Due to the strength of the appliedelectric field, the droplet may be strongly attracted to the right, intochannel 544. Similarly, in FIG. 9D, a dipole may be induced in droplet530 by applying an electric field 525 to the droplet using electrodes526 and 528, causing the droplet to be attracted into channel 542. Bycontrolling which electrodes are used to induce an electric field acrossdroplet 530, and/or the strength of the applied electric field, one ormore fluidic droplets within channel 540 may be sorted and/or split intotwo droplets, and each droplet may independently be sorted and/or split.

For example, highly monodisperse microcapsules containing a targetenzyme can be fused with highly monodisperse microcapsules each of whichcontain a different compound from a compound library. The fusedmicrocapsules flow along a microfluidic channel, allowing time for thecompounds to bind to the target enzyme. Each microcapsule is then fusedwith another microdroplet containing, for example, a fluorogenic enzymesubstrate. The rate of the enzymatic reaction is determined by measuringthe fluorescence of each microdroplet, ideally at multiple points(corresponding to different times).

Microcapsules can be optically tagged by, for example, incorporatingfluorochromes. In a preferred configuration, the microcapsules areoptically tagged by incorporating quantum dots: quantum dots of 6colours at 10 concentrations would allow the encoding of 10⁶microcapsules (Han et al., 2001). Microcapsules flowing in an orderedsequence in a microfluidic channel can be encoded (wholly or partially)by their sequence in the stream of microcapsules (positional encoding).

Microbeads, also known by those skilled in the art as microspheres,latex particles, beads, or minibeads, are available in diameters from 20nm to 1 mm and can be made from a variety of materials including silicaand a variety of polymers, copolymers and terpolymers includingpolystyrene (PS), polymethylmethacrylate (PMMA), polyvinyltoluene (PVT),styrene/butadiene (S/B) copolymer, and styrene/vinyltoluene (S/VT)copolymer (www.bangslabs.com). They are available with a variety ofsurface chemistries from hydrophobic surfaces (e.g. plain polystyrene),to very hydrophilic surfaces imparted by a wide variety of functionalsurface groups: aldehyde, aliphatic amine, amide, aromatic amine,carboxylic acid, chloromethyl, epoxy, hydrazide, hydroxyl, sulfonate andtosyl. The functional groups permit a wide range of covalent couplingreactions for stable or reversible attachment of compounds to themicrobead surface.

Microbeads can be optically tagged by, for example, incorporatingfluorochromes. For example, one hundred different bead sets have beencreated, each with a unique spectral address due to labelling withprecise ratios of red (>650 nm) and orange (585 nm) fluorochromes(Fulton et al., 1997) (www.luminex.com) and sets of up to 10⁶ beads canbe encoded by incorporating quantum dots of 10 intensities and 6 colours(Han et al., 2001).

The compounds can be connected to the microbeads either covalently ornon-covalently by a variety of means that will be familiar to thoseskilled in the art (see, for example, (Hermanson, 1996)).Advantageously, the compounds are attached via a cleavable linker. Avariety of such linkers are familiar to those skilled in the art (seefor example (Gordon and Balasubramanian, 1999)), including for example,linkers which can be cleaved photochemically and reversible covalentbonds which can be controlled by changing the pH (e.g. imines andacylhydrazones), by adjusting the oxido-reductive properties (e.g.disulphides), or using an external catalyst (e.g. cross-metathesis andtransamidation).

The method of the present invention permits the identification ofcompounds which modulate the activity of the target in a desired way inpools (libraries or repertoires) of compounds.

In a highly preferred application, the method of the present inventionis useful for screening libraries of compounds. The inventionaccordingly provides a method according to preceding aspects of theinvention, wherein the compounds are identified from a library ofcompounds.

The compounds identified according to the invention are advantageouslyof pharmacological or industrial interest, including activators orinhibitors of biological systems, such as cellular signal transductionmechanisms suitable for diagnostic and therapeutic applications. Inaddition the compounds identified according to the invention may benon-biological in nature. In a preferred aspect, therefore, theinvention permits the identification of clinically or industriallyuseful products. In a further aspect of the invention, there is provideda product when isolated by the method of the invention.

The selection of suitable encapsulation conditions is desirable.Depending on the complexity and size of the compound library to bescreened, it may be beneficial to set up the encapsulation proceduresuch that one or less than one secondary compound is formed permicrocapsule. This will provide the greatest power of resolution. Wherethe library is larger and/or more complex, however, this may beimpracticable; it may be preferable to form several secondary compoundstogether and rely on repeated application of the method of the inventionto identify the desired compound. A combination of encapsulationprocedures may be used to identify the desired compound.

Theoretical studies indicate that the larger the number of compoundscreated the more likely it is that a compound will be created with theproperties desired (see (Perelson and Oster, 1979) for a description ofhow this applies to repertoires of antibodies). It has also beenconfirmed practically that larger phage-antibody repertoires do indeedgive rise to more antibodies with better binding affinities than smallerrepertoires (Griffiths et al., 1994). To ensure that rare variants aregenerated and thus are capable of being identified, a large library sizeis desirable. Thus, the use of optimally small microcapsules isbeneficial.

The largest repertoires of compounds that can be screened in a singleexperiment to date, using two dimensional microarrays of 1 nl volumespots, is ˜10³ (Hergenrother et al., 2000). Using the present invention,at a microcapsule diameter of 2.6 mm (Tawfik and Griffiths, 1998), byforming a three-dimensional dispersion, a repertoire size of at least10¹¹ can be screened using 1 ml aqueous phase in a 20 ml emulsion.

In addition to the compounds, or microbeads coated with compounds,described above, the microcapsules according to the invention willcomprise further components required for the screening process to takeplace. They will comprise the target and a suitable buffer. A suitablebuffer will be one in which all of the desired components of thebiological system are active and will therefore depend upon therequirements of each specific reaction system. Buffers suitable forbiological and/or chemical reactions are known in the art and recipesprovided in various laboratory texts, such as (Sambrook and Russell,2001).

Other components of the system will comprise those necessary forassaying the activity of the target. These may for example comprisesubstrate(s) and cofactor(s) for a reaction catalysed by the target, andligand(s) bound by the target. They may also comprise other catalysts(including enzymes), substrates and cofactors for reactions coupled tothe activity of the target which allow for the activity of the target tobe detected.

(B) Screening Procedures

To screen compounds which bind to or modulate the activity of a target,the target is compartmentalised in microcapsules together with one ormore compounds or compound-coated microbeads. Advantageously eachmicrocapsule contains only a single sort of secondary compound, but manycopies thereof. Advantageously each microbead is coated with only asingle sort of compound, but many copies thereof. Advantageously thecompounds are connected to the microbeads via a cleavable linker,allowing them to be released from the microbeads in the compartments.Advantageously, each microcapsule or microbead is optically tagged toallow identification of the compounds contained within the microcapsuleof attached to the microbead.

(i) Screening for Binding

Compounds can be screened directly for binding to a target. In thisembodiment, if the compound is attached to a microbead and has affinityfor the target it will be bound by the target. At the end of thereaction, all of the microcapsules are combined, and all microbeadspooled together in one environment. Microbeads carrying compoundsexhibiting the desired binding can be selected by affinity purificationusing a molecule that specifically binds to, or reacts specificallywith, the target.

In an alternative embodiment, the target can be attached to microbeadsby a variety of means familiar to those skilled in the art (see forexample (Hermanson, 1996)). The compounds to be screened contain acommon feature—a tag. The compounds are released from the microbeads andif the compound has affinity for the target, it will bind to it. At theend of the reaction, all of the microcapsules are combined, and allmicrobeads pooled together in one environment. Microbeads carryingcompounds exhibiting the desired binding can be selected by affinitypurification using a molecule that specifically binds to, or reactsspecifically with, the “tag”.

In an alternative embodiment, microbeads may be screened on the basisthat the compound, which binds to the target, merely hides the ligandfrom, for example, further binding partners. In this eventuality, themicrobead, rather than being retained during an affinity purificationstep, may be selectively eluted whilst other microbeads are bound.

Sorting by affinity is dependent on the presence of two members of abinding pair in such conditions that binding may occur. Any binding pairmay be used for this purpose. As used herein, the term binding pairrefers to any pair of molecules capable of binding to one another.Examples of binding pairs that may be used in the present inventioninclude an antigen and an antibody or fragment thereof capable ofbinding the antigen, the biotin-avidin/streptavidin pair (Savage et al.,1994), a calcium-dependent binding polypeptide and ligand thereof (e.g.calmodulin and a calmodulin-binding peptide (Montigiani et al., 1996;Stofko et al., 1992), pairs of polypeptides which assemble to form aleucine zipper (Tripet et al., 1996), histidines (typicallyhexahistidine peptides) and chelated Cu²⁺, Zn²⁺ and Ni²⁺, (e.g. Ni—NTA;(Hochuli et al., 1987)), RNA-binding and DNA-binding proteins (Klug,1995) including those containing zinc-finger motifs (Klug and Schwabe,1995) and DNA methyltransferases (Anderson, 1993), and their nucleicacid binding sites.

In an alternative embodiment, compounds can be screened for binding to atarget using a change in the optical properties of the microcapsule orthe microbead.

The change in optical properties of the microcapsule or the microbeadafter binding of the compound to the target may be induced in a varietyof ways, including:

-   -   (1) the compound itself may have distinctive optical properties,        for example, it is fluorescent    -   (2) the optical properties of the compound may be modified on        binding to the target, for example, the fluorescence of the        compound is quenched or enhanced on binding (Voss, 1993; Masui        and Kuramitsu, 1998).    -   (3) the optical properties of the target may be modified on        binding of the compound, for example, the fluorescence of the        target is quenched or enhanced on binding (Guixe et al., 1998;        Qi and Grabowski, 1998)    -   (4) the optical properties of both target and compound are        modified on binding, for example, there can be a fluorescence        resonance energy transfer (FRET) from target to compound (or        vice versa) resulting in emission at the “acceptor” emission        wavelength when excitation is at the “donor” absorption        wavelength (Heim & Tsien, 1996; Mahajan et al., 1998; Miyawaki        et al., 1997).

The invention provides a method wherein a compound with the desiredactivity induces a change in the optical properties of the microcapsule,which enables the microcapsule containing the compound and themicrobeads contained therein to be identified, and optionally, sorted.

In an alternative embodiment, the invention provides a method whereinmicrobeads are analysed following pooling of the microcapsules into oneor more common compartments. In this embodiment, a compound having thedesired activity modifies the optical properties of the microbead whichcarried it (and which resides in the same microcapsule) to allow it tobe identified, and optionally, sorted.

In this embodiment, it is not necessary for binding of the compound tothe target to directly induce a change in optical properties.

In this embodiment, if the compound attached to the microbead hasaffinity for the target it will be bound by the target. At the end ofthe reaction, all of the microcapsules are combined, and all microbeadspooled together in one environment. Microbeads carrying compoundsexhibiting the desired binding can be identified by adding reagents thatspecifically bind to, or react specifically with, the target and therebyinduce a change in the optical properties of the microbeads allowingtheir identification. For example, a fluorescently-labelled anti-targetantibody can be used, or an anti-target antibody followed by a secondfluorescently labelled antibody which binds the first.

In an alternative embodiment, the target can be attached to themicrobeads by a variety of means familiar to those skilled in the art(see for example (Hermanson, 1996)). The compounds to be screenedcontain a common feature a tag. The compounds are released from themicrobeads and if the compound has affinity for the target, it will bindto it. At the end of the reaction, all of the microcapsules arecombined, and all microbeads pooled together in one environment.Microbeads carrying compounds exhibiting the desired binding can beidentified by adding reagents that specifically bind to, or reactspecifically with, the “tag” and thereby induce a change in the opticalproperties of the microbeads allowing their identification. For example,a fluorescently-labelled anti-“tag” antibody can be used, or ananti-“tag” antibody followed by a second fluorescently labelled antibodywhich binds the first.

In an alternative embodiment, microbeads may be identified on the basisthat the gene product, which binds to the ligand, merely hides theligand from, for example, further binding partners which would otherwisemodify the optical properties of the microbeads. In this case microbeadswith unmodified optical properties would be selected.

Fluorescence may be enhanced by the use of Tyramide Signal Amplification(TSA.™.) amplification to make the microbeads fluorescent (Sepp et al.,2002). This involves peroxidase (linked to another compound) binding tothe microbeads and catalysing the conversion of fluorescein-tyramine into a free radical form which then reacts (locally) with the microbeads.Methods for performing TSA are known in the art, and kits are availablecommercially from NEN.

TSA may be configured such that it results in a direct increase in thefluorescence of the microbeads, or such that a ligand is attached to themicrobeads which is bound by a second fluorescent molecule, or asequence of molecules, one or more of which is Fluorescent.

(ii) Screening for Regulation of Binding

In an alternative embodiment, the invention can be used to screencompounds which act to regulate a biochemical process. If the compoundactivates a binding activity of a target, a ligand for the target whichis activated can be attached to microbeads by a variety of meansfamiliar to those skilled in the art (see for example (Hermanson,1996)). At the end of the reaction, all of the microcapsules arecombined, and all microbeads pooled together in one environment.Microbeads carrying compounds exhibiting the desired binding can beselected by affinity purification using a molecule that specificallybinds to, or reacts specifically with, the target.

In an alternative embodiment, microbeads may be screened on the basisthat the compound inhibits the binding activity of a target. In thiseventuality, the microbead, rather than being retained during anaffinity purification step, may be selectively eluted whilst othermicrobeads are bound.

In an alternative embodiment, compounds can be screened for the abilityto modulates a binding activity of a target using a change in theoptical properties of the microcapsule or the microbead.

The change in optical properties of the microcapsule or the microbeadafter binding of the target to its ligand may be induced in a variety ofways, including:

-   -   (1) the ligand itself may have distinctive optical properties,        for example, it is fluorescent    -   (2) the optical properties of the ligand may be modified on        binding to the target, for example, the fluorescence of the        ligand is quenched or enhanced on binding (Voss, 1993; Masui and        Kuramitsu, 1998).    -   (3) the optical properties of the target may be modified on        binding of the ligand, for example, the fluorescence of the        target is quenched or enhanced on binding (Guixe et al., 1998;        Qi and Grabowski, 1998)    -   (4) the optical properties of both target and ligand are        modified on binding, for example, there can be a fluorescence        resonance energy transfer (FRET) from target to ligand (or vice        versa) resulting in emission at the “acceptor” emission        wavelength when excitation is at the “donor” absorption        wavelength (Heim & Tsien, 1996; Mahajan et al., 1998; Miyawaki        et al., 1997).

The invention provides a method wherein a compound with the desiredactivity induces a change in the optical properties of the microcapsule,which enables the microcapsule containing the compound and themicrobeads contained therein to be identified, and optionally, sorted.

In an alternative embodiment, the invention provides a method whereinmicrobeads are analysed following pooling of the microcapsules into oneor more common compartments. In this embodiment, a compound having thedesired activity modifies the optical properties of the microbead whichcarried it (and which resides in the same microcapsule) to allow it tobe identified, and optionally, sorted.

In this embodiment, it is not necessary for binding of the target to theligand to directly induce a change in optical properties.

In this embodiment, if a ligand attached to the microbead has affinityfor the target it will be bound by the target. At the end of thereaction, all of the microcapsules are combined, and all microbeadspooled together in one environment. Microbeads carrying compounds whichmodulate the binding activity can be identified by adding reagents thatspecifically bind to, or react specifically with, the target and therebyinduce a change in the optical properties of the microbeads allowingtheir identification. For example, a fluorescently-labelled anti-targetantibody can be used, or an anti-target antibody followed by a secondfluorescently labelled antibody which binds the first.

In an alternative embodiment, the target can be attached to themicrobeads by a variety of means familiar to those skilled in the art(see for example (Hermanson, 1996)). The ligand to be screened containsa feature—a tag. At the end of the reaction, all of the microcapsulesare combined, and all microbeads pooled together in one environment.Microbeads carrying compounds which modulate binding can be identifiedby adding reagents that specifically bind to, or react specificallywith, the “tag” and thereby induce a change in the optical properties ofthe microbeads allowing their identification. For example, afluorescently-labelled anti-“tag” antibody can be used, or an anti-“tag”antibody followed by a second fluorescently labelled antibody whichbinds the first.

Fluorescence may be enhanced by the use of Tyramide Signal Amplification(TSA.™.) amplification to make the microbeads fluorescent (Sepp et al.,2002), as above.

(iii) Screening for Regulation of Catalysis

In an alternative embodiment, the invention provides a method wherein acompound with the desired activity induces a change in the opticalproperties of the microcapsule, which enables the microcapsulecontaining the compound and, optionally, the microbeads containedtherein to be identified, and optionally, sorted. The optical propertiesof microcapsules can be modified by either:

-   -   (a) the substrate and product of the regulated reaction having        different optical properties (many fluorogenic enzyme substrates        are available commercially, see for example (Haugland, 1996 and        www.probes.com) including substrates for glycosidases,        phosphatases, peptidases and proteases, or    -   (b) the presence of reagents which specifically bind to, or        react with, the product (or substrate) of the regulated reaction        in the microcapsule and which thereby induce a change in the        optical properties of the microcapsules allowing their        identification.

A wide range of assays for screening libraries of compounds for thosewhich modulate the activity of a target are based on detecting changesin optical properties and can be used to screen compounds according tothis invention. Such assays are well known to those skilled in the art(see for example Haugland, 1996 and www.probes.com).

Alternatively, selection may be performed indirectly by coupling a firstreaction to subsequent reactions that takes place in the samemicrocapsule. There are two general ways in which this may be performed.First, the product of the first reaction could be reacted with, or boundby, a molecule which does not react with the substrate(s) of the firstreaction. A second, coupled reaction will only proceed in the presenceof the product of the first reaction. A regulatory compound can then beidentified by the properties of the product or substrate of the secondreaction.

Alternatively, the product of the reaction being selected may be thesubstrate or cofactor for a second enzyme-catalysed reaction. The enzymeto catalyse the second reaction can be incorporated in the reactionmixture prior to microencapsulation. Only when the first reactionproceeds will the coupled enzyme generate an identifiable product.

This concept of coupling can be elaborated to incorporate multipleenzymes, each using as a substrate the product of the previous reaction.This allows for selection of regulators of enzymes that will not reactwith an immobilised substrate. It can also be designed to give increasedsensitivity by signal amplification if a product of one reaction is acatalyst or a cofactor for a second reaction or series of reactionsleading to a selectable product (for example, see (Johannsson, 1991;Johannsson and Bates, 1988). Furthermore an enzyme cascade system can bebased on the production of an activator for an enzyme or the destructionof an enzyme inhibitor (see (Mize et al., 1989)). Coupling also has theadvantage that a common screening system can be used for a whole groupof enzymes which generate the same product and allows for the selectionof regulation of complicated multi-step chemical transformations andpathways.

In an alternative embodiment, if the target is itself an enzyme, orregulates a biochemical process which is enzymatic, the microbead ineach microcapsule may be coated with the substrate for the enzymaticreaction. The regulatory compound will determine the extent to which thesubstrate is converted into the product. At the end of the reaction themicrobead is physically linked to the product of the catalysed reaction.When the microcapsules are combined and the reactants pooled, microbeadswhich were coated with activator compounds can be identified by anyproperty specific to the product. If an inhibitor is desired, selectioncan be for a chemical property specific to the substrate of theregulated reaction.

It may also be desirable, in some cases, for the substrate not to beattached to the microbead. In this case the substrate would contain aninactive “tag” that requires a further step to activate it such asphotoactivation (e.g. of a “caged” biotin analogue, (Pirrung and Huang,1996; Sundberg et al., 1995)). After convertion of the substrate toproduct the “tag” is activated and the “tagged” substrate and/or productbound by a tag-binding molecule (e.g. avidin or streptavidin) attachedto the microbead. The ratio of substrate to product attached to thenucleic acid via the “tag” will therefore reflect the ratio of thesubstrate and product in solution. A substrate tagged with caged biotinhas been used to select for genes encoding enzymes withphosphotriesterase activity using a procedure based oncompartmentalisation in microcapsules (Griffiths and Tawfik, 2003). Thephosphotriesterase substrate was hydrolysed in solution in microcapsulescontaining active enzyme molecules, and after the reaction wascompleted, the caging group was released by irradiation to allow theproduct to bind, via the biotin moiety, to microbeads to which the geneencoding the enzyme was attached.

After the microbeads and the contents of the microcapsules are combined,those microbeads coated with regulators can be selected by affinitypurification using a molecule (e.g. an antibody) that binds specificallyto the product or substrate as appropriate.

In an alternative embodiment, the invention provides a method whereinthe microbeads are analysed following pooling of the microcapsules intoone or more common compartments. Microbeads coated with regulatorcompounds can be identified using changes in optical properties of themicrobeads. The optical properties of microbeads with product (orsubstrate) attached can be modified by either:

-   -   (1) the product-microbead complex having characteristic optical        properties not found in the substrate-microbead complex, due to,        for example;        -   (a) the substrate and product having different optical            properties (many fluorogenic enzyme substrates are available            commercially (see for example Haugland, 1996 and            www.probes.com) including substrates for glycosidases,            phosphatases, peptidases and proteases, or        -   (b) the substrate and product having similar optical            properties, but only the product, and not the substrate            binds to, or reacts with, the microbead;    -   (2) adding reagents which specifically bind to, or react with,        the product (or substrate) and which thereby induce a change in        the optical properties of the microbeads allowing their        identification (these reagents can be added before or after        breaking the microcapsules and pooling the microbeads). The        reagents;        -   (a) bind specifically to, or react specifically with, the            product, and not the substrate, (or vice versa) if both            substrate and product are attached to the microbeads, or        -   (b) optionally bind both substrate and product if only the            product, and not the substrate binds to, or reacts with, the            microbeads (or vice versa).

In this scenario, the substrate (or one of the substrates) can bepresent in each microcapsule unlinked to the microbead, but has amolecular “tag” (for example biotin, DIG or DNP or a fluorescent group).When the regulated enzyme converts the substrate to product, the productretains the “tag” and is then captured in the microcapsule by theproduct-specific antibody. When all reactions are stopped and themicrocapsules are combined, these microbeads will be “tagged” and mayalready have changed optical properties, for example, if the “tag” was afluorescent group. Alternatively, a change in optical properties of“tagged” microbeads can be induced by adding a fluorescently labelledligand which binds the “tag” (for example fluorescently-labelledavidin/streptavidin, an anti-“tag” antibody which is fluorescent, or anon-fluorescent anti-“tag” antibody which can be detected by a secondfluorescently-labelled antibody).

(iv) Screening for Compound Specificity/Selectivity

Compounds with specificity or selectivity for certain targets and notothers can be specifically identified by carrying out a positive screenfor regulation of a reaction using one substrate and a negative screenfor regulation of a reaction with another substrate.

For example, two substrates, specific for two different target enzymes,are each labelled with different fluorogenic moieties. Each targetenzymes catalyse the generation of a product with with a differentfluorescence spectrum resulting in different optical properties of themicrocapsules depending on the specificity of the compound for twotargets.

(v) Screening Using Cells

In the current drug discovery paradigm, validated recombinant targetsform the basis of in vitro high-throughput screening (HTS) assays.Isolated proteins cannot, however, be regarded as representative ofcomplex biological systems; hence, cell-based systems can providegreater confidence in compound activity in an intact biological system.A wide range of cell-based assays for drug leads are known to thoseskilled in the art. Cells can be compartmentalised in microcapsules,such as the aqeous microdroplets of a water-in-oil emulsion (Ghadessy,2001). The effect of a compound(s) on a target can be determined bycompartmentalising a cell (or cells) in a microcapsule together with acompound(s) and using an appropriate cell-based assay to identify thosecompartments containing compounds with the desired effect on thecell(s). The use of water-in-fluorocarbon emulsions may be particularlyadvantageous: the high gas dissolving capacity of fluorocarbons cansupport the exchange of respiratory gases and has been reported to bebeneficial to cell culture systems (Lowe, 2002).

(vi) Flow Cytometry

(vi) Flow Analysis and Sorting

In a preferred embodiment of the invention the microcapsules ormicrobeads will be analysed and, optionally, sorted by flow cytometry.Many formats of microcapsule can be analysed and, optionally, sorteddirectly u sing flow cytometry.

In a highly preferred embodiment, microfluidic devices for flow analysisand, optionally, flow sorting (Fu, 2002) of microdroplets and microbeadswill be used.

A variety of optical properties can be used for analysis and to triggersorting, including light scattering (Kerker, 1983) and fluorescencepolarisation (Rolland et al., 1985). In a highly preferred embodimentthe difference in optical properties of the microcapsules or microbeadswill be a difference in fluorescence and, if required, the microcapsulesor microbeads will be sorted using a microfluidic or conventionalfluorescence activated cell sorter (Norman, 1980; Mackenzie and Pinder,1986), or similar device. Flow cytometry has a series of advantages:

-   -   (1) fluorescence activated cell sorting equipment from        established manufacturers (e.g. Becton-Dickinson, Coulter,        Cytomation) allows the analysis and sorting at up to 100,000        microcapsules or microbeads s−1.    -   (2) the fluorescence signal from each microcapsule or microbead        corresponds tightly to the number of fluorescent molecules        present. As little as few hundred fluorescent molecules per        microcapsules or microbeads can be quantitatively detected;    -   (3) the wide dynamic range of the fluorescence detectors        (typically 4 log units) allows easy setting of the stringency of        the sorting procedure, thus allowing the recovery of the optimal        number microcapsules or microbeads from the starting pool (the        gates can be set to separate microcapsules or microbeads with        small differences in fluorescence or to only separate out        microcapsules or microbeads with large differences in        fluorescence, dependant on the selection being performed);    -   (4) fluorescence-activated cell sorting equipment can perform        simultaneous excitation and detection at multiple wavelengths        (Shapiro, 1995) allowing positive and negative selections to be        performed simultaneously by monitoring the labelling of the        microcapsules or microbeads with two to thirteen (or more)        fluorescent markers, for example, if substrates for two        alternative targets are labelled with different fluorescent tags        the microcapsules or microbeads can labelled with different        fluorophores dependent on the target regulated.

If the microcapsules or microbeads are optically tagged, flow cytometrycan also be used to identify the compound or compounds in themicrocapsule or coated on the microbeads (see below). Optical taggingcan also be used to identify the concentration of the compound in themicrocapsule (if more than one concentration is used in a singleexperiment) or the number of compound molecules coated on a microbead(if more than one coating density is used in a single experiment).Furthermore, optical tagging can be used to identify the target in amicrocapsule (if more than one target is used in a single experiment).This analysis can be performed simultaneously with measuring activity,after sorting of microcapsules containing microbeads, or after sortingof the microbeads.

(vii) Microcapsule Identification and Sorting

The invention provides for the identification and, optionally, thesorting of intact microcapsules where this is enabled by the sortingtechniques being employed. Microcapsules may be identified and,optionally, sorted as such when the change induced by the desiredcompound either occurs or manifests itself at the surface of themicrocapsule or is detectable from outside the microcapsule. The changemay be caused by the direct action of the compound, or indirect, inwhich a series of reactions, one or more of which involve the compoundhaving the desired activity leads to the change. For example, where themicrocapsule is a membranous microcapsule, the microcapsule may be soconfigured that a component or components of the biochemical systemcomprising the target are displayed at its surface and thus accessibleto reagents which can detect changes in the biochemical system regulatedby the compound on the microbead within the microcapsule.

In a preferred aspect of the invention, however, microcapsuleidentification and, optionally, sorting relies on a change in theoptical properties of the microcapsule, for example absorption oremission characteristics thereof, for example alteration in the opticalproperties of the microcapsule resulting from a reaction leading tochanges in absorbance, luminescence, phosphorescence or fluorescenceassociated with the microcapsule. All such properties are included inthe term “optical”. In such a case, microcapsules can be identified and,optionally, sorted by luminescence, fluorescence or phosphorescenceactivated sorting. In a highly preferred embodiment, flow cytometry isemployed to analyse and, optionally, sort microcapsules containingcompounds having a desired activity which result in the production of afluorescent molecule in the microcapsule.

The methods of the current invention allow reagents to be mixed rapidly(in <2 ms), hence a spatially-resolved optical image of microcapsules inmicrofluidic network allows time resolved measurements of the reactionsin each microcapsule. Microcapsules can, optionally, be separated usinga microfluidic flow sorter to allow recovery and further analysis ormanipulation of the molecules they contain. Advantageously, the flowsorter would be an electronic flow sorting device. Such a sorting devicecan be integrated directly on the microfluidic device, and can useelectronic means to sort the microcapsules. Optical detection, alsointegrated directly on the microfluidic device, can be used to screenthe microcapsules to trigger the sorting. Other means of control of themicrocapsules, in addition to charge, can also be incorporated onto themicrofluidic device.

In an alternative embodiment, a change in microcapsule fluorescence,when identified, is used to trigger the modification of the microbeadwithin the compartment. In a preferred aspect of the invention,microcapsule identification relies on a change in the optical propertiesof the microcapsule resulting from a reaction leading to luminescence,phosphorescence or fluorescence within the microcapsule. Modification ofthe microbead within the microcapsules would be triggered byidentification of luminescence, phosphorescence or fluorescence. Forexample, identification of luminescence, phosphorescence or fluorescencecan trigger bombardment of the compartment with photons (or otherparticles or waves) which leads to modification of the microbead ormolecules attached to it. A similar procedure has been describedpreviously for the rapid sorting of cells (Keij et al., 1994).Modification of the microbead may result, for example, from coupling amolecular “tag”, caged by a photolabile protecting group to themicrobeads: bombardment with photons of an appropriate wavelength leadsto the removal of the cage. Afterwards, all microcapsules are combinedand the microbeads pooled together in one environment. Microbeads coatedwith compounds exhibiting the desired activity can be selected byaffinity purification using a molecule that specifically binds to, orreacts specifically with, the “tag”.

(C) Compounds Libraries

(i) Primary Compound Libraries

Libraries of primary compounds can be obtained from a variety ofcommercial sources. The compounds in the library can be made by avariety of means well known to those skilled in the art. Optionally,compound libraries can be made by combinatorial synthesis usingspatially resolved parallel synthesis or using split synthesis,optionally to generate one-bead-one-compound libraries. The compoundscan, optionally, be synthesised on beads. These beads can becompartmentalised in microcapsules directly or the compounds releasedbefore compartmentalisation.

Advantageously, only a single type of compound, but multiple copiesthereof is present in each microcapsule.

The compounds can, optionally, be connected to microbeads eithercovalently or non-covalently by a variety of means that will be familiarto those skilled in the art (see, for example, (Hermanson, 1996)).

Microbeads are available with a variety of surface chemistries fromhydrophobic surfaces (e.g. plain polystyrene), to very hydrophilicsurfaces imparted by a wide variety of functional surface groups:aldehyde, aliphatic amine, amide, aromatic amine, carboxylic acid,chloromethyl, epoxy, hydrazide, hydroxyl, sulfonate and tosyl. Thefunctional groups permit a wide range of covalent coupling reactions,well known to those skilled in the art, for stable or reversibleattachment of compounds to the microbead surface.

Advantageously, the compounds are attached to the microbeads via acleavable linker. A variety of such linkers are familiar to thoseskilled in the art (see for example (Gordon and Balasubramanian, 1999)),including for example, linkers which can be cleaved photochemically andreversible covalent bonds which can be controlled by changing the pH(e.g. imines and acylhydrazones), by adjusting the oxido-reductiveproperties (e.g. disulphides), or using an external catalyst (e.g.cross-metathesis and transamidation).

Advantageously, only a single type of compound, but multiple copiesthereof is attached to each bead.

(ii) Secondary Compound Libraries

Secondary compound libraries are created by reactions between primarycompounds in microcapsules. Secondary compounds can be created by avariety of two component, and multi-component reactions well known tothose skilled in the art (Armstrong et al., 1996; Domling, 2002; Domlingand Ugi, 2000; Ramstrom and Lehn, 2002).

To form secondary compound libraries by a two-component reaction, twosets of compounds are compartmentalised in microcapsules such that manycompartments contain two or more compounds. Advantageously, the modalnumber of compounds per microcapsule is two. Advantageously, themicrocapsules contain at least one type of compound from each set ofcompounds. Advantageously, the microcapsules contain one type ofcompound from each set of compounds. The secondary compounds are formedby chemical reactions between primary compounds from different sets. Thesecondary compound may be the result of a covalent or non-covalentreaction between the primary compounds.

A variety of chemistries, familiar to those skilled in the art, aresuitable to form secondary compounds in two-component reactions. Forexample, reversible covalent bonds which can be controlled by changingthe pH (e.g. imines and acylhydrazones), by adjusting theoxido-reductive properties (e.g. disulphides), or using an externalcatalyst (e.g. cross-metathesis and transamidation), can be used(Ramstrom and Lehn, 2002).

In a further embodiment, the method can also be used to create secondarycompound libraries using three-component, four-component and higherorder multi-component reactions. Three, four or more sets of compounds(as appropriate) are compartmentalised in microcapsules. The compoundsare compartmentalised in microcapsules such that many compartmentscontain multiple compounds. Advantageously, the modal number ofcompounds per microcapsule is equal to the number of components in thereaction. Advantageously, the microcapsules contain at least one type ofcompound from each set of compounds. Advantageously, the microcapsulescontain one type of compound from each set of compounds. The secondarycompounds are formed by chemical reactions between primary compoundsfrom different sets. The secondary compound may be the result ofcovalent or non-covalent reactions between the primary compounds.

Examples of suitable multi-component reactions are the Strecker,Hantzsch, Biginelli, Mannich, Passerini, Bucherer-Bergs and Pauson-Khandthree-component reactions and the Ugi four-component reaction (Armstronget al., 1996; Domling, 2002; Domling and Ugi, 2000).

Secondary compound libraries may also be built using a scaffold moleculewhich is common to all the secondary compounds (Ramstrom and Lehn,2002). This scaffold molecule may be compartmentalised intomicrocapsules together with the other primary compounds.

In a further embodiment, to form secondary compound libraries by atwo-component reaction, two sets of compounds are attached tomicrobeads, advantageously to give only a single type of molecule permicrobead. The microbeads are compartmentalised in microcapsules suchthat many compartments contain two or more microbeads. Advantageously,the modal number of beads per microcapsule is two. The compoundscomprising at least one of the two sets are released from themicrobeads. The secondary compounds are formed by chemical reactionsbetween primary compounds from different sets. The secondary compoundmay be the result of a covalent or non-covalent reaction between theprimary compounds.

In a further embodiment, the method can also be used to create secondarycompound libraries using three-component, four-component and higherorder multi-component reactions. Three, four or more sets of compounds(as appropriate) are attached to microbeads, advantageously to give onlya single type of molecule per microbead. The microbeads arecompartmentalised in microcapsules such that many compartments containmultiple microbeads. Advantageously, the modal number of beads permicrocapsule is equal to the number of components in the reaction. Thecompounds comprising either all, or all bar one; of the sets arereleased from the microbeads. The secondary compounds are formed bychemical reactions between primary compounds from different sets. Thesecondary compound may be the result of covalent or non-covalentreactions between the primary compounds.

Advantageously, the same reversible covalent bond can used to couple theprimary compound to the microbead as is used to form the secondarycompound.

Secondary compound libraries may also be built using a scaffold moleculewhich is common to all the secondary compounds (Ramstrom and Lehn,2002). This scaffold molecule may be compartmentalised intomicrocapsules together with the microbeads.

(D) Identification of Compounds

The compounds in microcapsules or on microbeads can be identified in avariety of ways. If the identified microcapsules are sorted (e.g. byusing a fluorescence activated cell sorter—FACS) the compounds can beidentified by direct analysis, for example by mass-spectroscopy. If thecompounds remain attached to beads isolated as a result of selection(for example by affinity purification) or sorting (for example using aFACS) they can, also be identified by direct analysis, for example bymass-spectroscopy. The microcapsules or beads can also be tagged by avariety of means well known to those skilled in the art and the tag usedto identify the compound attached to the beads (Czamik, 1997). Chemical,spectrometric, electronic, and physical methods to encode the compoundsmay all be used. In a preferred embodiment microcapsules or beads havedifferent optical properties and are thereby optically encoded. In apreferred embodiment encoding is based on microcapsules or beads havingdifferent fluorescence properties. In a highly preferred embodiment themicrocapsules or beads are encoded using fluorescent quantum dotspresent at different concentrations in the microcapsule or bead (Han,2001). Microcapsules flowing in an ordered sequence in a microfluidicchannel can also be encoded (wholly or partially) by their sequence inthe stream of microcapsules (positional encoding).

Advantageously, each compounds is present in different microcapsules atdifferent concentrations (typically at concentrations varying from mM tonM) allowing the generation of a dose-response curve. Fusingmicrocapsules to give all possible permutations of several differentsubstrate concentrations and compound concentrations would allow thedetermination of the mode of inhibition (e.g. competitive,noncompetitive, uncompetitive or mixed inhibition) and inhibitionconstant (K_(i)) of an inhibitory compound. The concentration of thecompounds in the microcapsules can be determined by, for example,optical encoding or positional encoding of the microcapsules ormicrobeads as above.

(E) Identification of Targets

Advantageously, multiple different targets can be compartmentalised inmicrocapsules such that each microcapsule contains multiple copies ofthe same target. For example, multiple protein kinases, or multiplepolymorphic variants of a single target, can be compartmentalised toallow the specificity of compounds to be determined. The identity of thetarget in a micro capsule can be determined by, for example, opticalencoding or positional encoding of the microcapsules or microbeads asabove.

Expressed in an alternative manner, there is provided a method for thesynthesis and identification of compounds which bind to a targetcomponent of a biochemical system or modulate the activity of thetarget, comprising the steps of.

-   -   (a) compartmentalising two or more sets of primary compounds        into microcapsules together with the target such that many        compartments contain two or more primary compounds;    -   (b) forming secondary compounds in the microcapsules by chemical        reactions between primary compounds from different sets; and    -   (c) identifying subsets of primary compounds which react to form        secondary compounds which bind to or modulate the activity of        the target.

There is also provided a method for the synthesis and identification ofcompounds which bind to a target component of a biochemical system ormodulate the activity of the target, comprising the steps of:

-   -   (1) attaching two or more sets of primary compounds onto        microbeads;    -   (2) compartmentalising the microbeads into microcapsules        together with the target such that many compartments contain two        or more microbeads;    -   (3) releasing the primary compounds from the microbeads;    -   (4) forming secondary compounds in the microcapsules by chemical        reactions between primary compounds from different sets; and    -   (5) identifying subsets of primary compounds which react to form        secondary compounds which bind to or modulate the activity of        the target.

If the primary compounds react, not only with other primary compounds inthe same compartment, but also with other microbeads in the compartment,the primary compounds which react together to form a secondary compoundcan be identified by direct analysis of the compounds present on amicrobeads isolated as a result of selection or sorting. For example, ifthe primary compounds are linked to the beads via a disulphide bond whenthey are released in the compartment the primary compounds will reactboth with each other to form a secondary compound and with thesulphydryl groups on the beads. Hence, if two beads areco-compartmentalised, each bead will end up carrying both primarycompounds. After isolation of these beads both primary compounds whichreacted to form the secondary compound can be identified.

(F) Rapid Mixing of Reagents in Microcapsules

Advantageously, after fusion of microcpasules, the reagents contained inthe fused microcapsule can be mixed rapidly using chaotic advection. Bypassing the droplets through channels that disrupt the laminar flowlines of the fluid within the droplets, their contents can be rapidlymixed, fully initiating any chemical reactions.

(G) Sensing Microcapsule Characteristics

In certain aspects of the invention, sensors are provided that can senseand/or determine one or more characteristics of the fluidic droplets,and/or a characteristic of a portion of the fluidic system containingthe fluidic droplet (e.g., the liquid surrounding the fluidic droplet)in such a manner as to allow the determination of one or morecharacteristics of the fluidic droplets. Characteristics determinablewith respect to the droplet and usable in the invention can beidentified by those of ordinary skill in the art. Non-limiting examplesof such characteristics include fluorescence, spectroscopy (e.g.,optical, infrared, ultraviolet, etc.), radioactivity, mass, volume,density, temperature, viscosity, pH, concentration of a substance, suchas a biological substance (e.g., a protein, a nucleic acid, etc.), orthe like.

In some cases, the sensor may be connected to a processor, which inturn, cause an operation to be performed on the fluidic droplet, forexample, by sorting the droplet, adding or removing electric charge fromthe droplet, fusing the droplet with another droplet, splitting thedroplet, causing mixing to occur within the droplet, etc., for example,as previously described. For instance, in response to a sensormeasurement of a fluidic droplet, a processor may cause the fluidicdroplet to be split, merged with a second fluidic droplet, sorted etc.

One or more sensors and/or processors may be positioned to be in sensingcommunication with the fluidic droplet. “Sensing communication,” as usedherein, means that the sensor may be positioned anywhere such that thefluidic droplet within the fluidic system (e.g., within a channel),and/or a portion of the fluidic system containing the fluidic dropletmay be sensed and/or determined in some fashion. For example, the sensormay be in sensing communication with the fluidic droplet and/or theportion of the fluidic system containing the fluidic droplet fluidly,optically or visually, thermally, pneumatically, electronically, or thelike. The sensor can be positioned proximate the fluidic system, forexample, embedded within or integrally connected to a wall of a channel,or positioned separately from the fluidic system but with physical,electrical, and/or optical communication with the fluidic system so asto be able to sense and/or determine the fluidic droplet and/or aportion of the fluidic system containing the fluidic droplet (e.g., achannel or a microchannel, a liquid containing the fluidic droplet,etc.). For example, a sensor may be free of any physical connection witha channel containing a droplet, but may be positioned so as to detectelectromagnetic radiation arising from the droplet or the fluidicsystem, such as infrared, ultraviolet, or visible light. Theelectromagnetic radiation may be produced by the droplet, and/or mayarise from other portions of the fluidic system (or Externally of thefluidic system) and interact with the fluidic droplet and/or the portionof the fluidic system containing the fluidic droplet in such as a manneras to indicate one or more characteristics of the fluidic droplet, forexample, through absorption, reflection, diffraction, refraction,fluorescence, phosphorescence, changes in polarity, phase changes,changes with respect to time, etc. As an example, a laser may bedirected towards the fluidic droplet and/or the liquid surrounding thefluidic droplet, and the fluorescence of the fluidic droplet and/or thesurrounding liquid may be determined. “Sensing communication,” as usedherein may also be direct or indirect. As an example, light from thefluidic droplet may be directed to a sensor, or directed first through afiber optic system, a waveguide, etc., before being directed to asensor.

Non-limiting examples of sensors useful in the invention include opticalor electromagnetically-based systems. For example, the sensor may be afluorescence sensor (e.g., stimulated by a laser), a microscopy system(which may include a camera or other recording device), or the like. Asanother example, the sensor may be an electronic sensor, e.g., a sensorable to determine an electric field or other electrical characteristic.For example, the sensor may detect capacitance, inductance, etc., of afluidic droplet and/or the portion of the fluidic system containing thefluidic droplet.

As used herein, a “processor” or a “microprocessor” is any component ordevice able to receive a signal from one or more sensors, store thesignal, and/or direct one or more responses (e.g., as described above),for example, by using a mathematical formula or an electronic orcomputational circuit. The signal may be any suitable signal indicativeof the environmental factor determined by the sensor, for example apneumatic signal, an electronic signal, an optical signal, a mechanicalsignal, etc.

(H) Materials

A variety of materials and methods, according to certain aspects of theinvention, can be used to form any of the above-described components ofthe microfluidic systems and devices of the invention. In some cases,the various materials selected lend themselves to various methods. Forexample, various components of the invention can be formed from solidmaterials, in which the channels can be formed via micromachining, filmdeposition processes such as spin coating and chemical vapor deposition,laser fabrication, photolithographic techniques, etching methodsincluding wet chemical or plasma processes, and the like. See, forexample, Scientific American, 248:44-55, 1983 (Angell, et al). In oneembodiment, at least a portion of the fluidic system is formed ofsilicon by etching features in a silicon chip. Technologies for preciseand efficient fabrication of various fluidic systems and devices of theinvention from silicon are known. In another embodiment, variouscomponents of the systems and devices of the invention can be formed ofa polymer, for example, an elastomeric polymer such aspolydimethylsiloxane (“PDMS”), polytetrafluoroethylene (“PTFE” orTeflon.®.), or the like.

Different components can be fabricated of different materials. Forexample, a base portion including a bottom wall and side walls can befabricated from an opaque material such as silicon or PDMS, and a topportion can be fabricated from a transparent or at least partiallytransparent material, such as glass or a transparent polymer, forobservation and/or control of the fluidic process. Components can becoated so as to expose a desired chemical functionality to fluids thatcontact interior channel walls, where the base supporting material doesnot have a precise, desired functionality. For example, components canbe fabricated as illustrated, with interior channel walls coated withanother material. Material used to fabricate various components of thesystems and devices of the invention, e.g., materials used to coatinterior walls of fluid channels, may desirably be selected from amongthose materials that will not adversely affect or be affected by fluidflowing through the fluidic system, e.g., material(s) that is chemicallyinert in the presence of fluids to be used within the device.

In one embodiment, various components of the invention are fabricatedfrom polymeric and/or flexible and/or elastomeric materials, and can beconveniently formed of a hardenable fluid, facilitating fabrication viamolding (e.g. replica molding, injection molding, cast molding, etc.).The hardenable fluid can be essentially any fluid that can be induced tosolidify, or that spontaneously solidifies, into a solid capable ofcontaining and/or transporting fluids contemplated for use in and withthe fluidic network. In one embodiment, the hardenable fluid comprises apolymeric liquid or a liquid polymeric precursor (i.e. a “prepolymer”).Suitable polymeric liquids can include, for example, thermoplasticpolymers, thermoset polymers, or mixture of such polymers heated abovetheir melting point. As another example, a suitable polymeric liquid mayinclude a solution of one or more polymers in a suitable solvent, whichsolution forms a solid polymeric material upon removal of the solvent,for example, by evaporation. Such polymeric materials, which can besolidified from, for example, a melt state or by solvent evaporation,are well known to those of ordinary skill in the art. A variety ofpolymeric materials, many of which are elastomeric, are suitable, andare also suitable for forming molds or mold masters, for embodimentswhere one or both of the mold masters is composed of an elastomericmaterial. A non-limiting list of examples of such polymers includespolymers of the general classes of silicone polymers, epoxy polymers,and acrylate polymers. Epoxy polymers are characterized by the presenceof a three-membered cyclic ether group commonly referred to as an epoxygroup, 1,2-epoxide, or oxirane. For example, diglycidyl ethers ofbisphenol A can be used, in addition to compounds based on aromaticamine, triazine, and cycloaliphatic backbones. Another example includesthe well-known Novolac polymers. Non-limiting examples of siliconeelastomers suitable for use according to the invention include thoseformed from precursors including the chlorosilanes such asmethylchlorosilanes, ethylchlorosilanes, phenylchlorosilanes, etc.

Silicone polymers are preferred in one set of embodiments, for example,the silicone elastomer polydimethylsiloxane. Non-limiting examples ofPDMS polymers include those sold under the trademark Sylgard by DowChemical Co., Midland, Mich., and particularly Sylgard 182, Sylgard 184,and Sylgard 186. Silicone polymers including PDMS have severalbeneficial properties simplifying fabrication of the microfluidicstructures of the invention. For instance, such materials areinexpensive, readily available, and can be solidified from aprepolymeric liquid via curing with heat. For example, PDMSs aretypically curable by exposure of the prepolymeric liquid to temperaturesof about, for example, about 65° C. to about 75° C. for exposure timesof, for example, about an hour. Also, silicone polymers, such as PDMS,can be elastomeric and thus may be useful for forming very smallfeatures with relatively high aspect ratios, necessary in certainembodiments of the invention. Flexible (e.g., elastomeric) molds ormasters can be advantageous in this regard.

One advantage of forming structures such as microfluidic structures ofthe invention from silicone polymers, such as PDMS, is the ability ofsuch polymers to be oxidized, for example by exposure to anoxygen-containing plasma such as an air plasma, so that the oxidizedstructures contain, at their surface, chemical groups capable ofcross-linking to other oxidized silicone polymer surfaces or to theoxidized surfaces of a variety of other polymeric and non-polymericmaterials. Thus, components can be fabricated and then oxidized andessentially irreversibly sealed to other silicone polymer surfaces, orto the surfaces of other substrates reactive with the oxidized siliconepolymer surfaces, without the need for separate adhesives or othersealing means. In most cases, sealing can be completed simply bycontacting an oxidized silicone surface to another surface without theneed to apply auxiliary pressure to form the seal. That is, thepre-oxidized silicone surface acts as a contact adhesive againstsuitable mating surfaces. Specifically, in addition to beingirreversibly sealable to itself, oxidized silicone such as oxidized PDMScan also be sealed irreversibly to a range of oxidized materials otherthan itself including, for example, glass, silicon, silicon oxide,quartz, silicon nitride, polyethylene, polystyrene, glassy carbon, andepoxy polymers, which have been oxidized in a similar fashion to thePDMS surface (for example, via exposure to an oxygen-containing plasma).Oxidation and sealing methods useful in the context of the presentinvention, as well as overall molding techniques, are described in theart, for example, in an article entitled “Rapid Prototyping ofMicrofluidic Systems and Polydimethylsiloxane,” Anal. Chem., 70:474-480,1998 (Duffy et al.), incorporated herein by reference.

Another advantage to forming microfluidic structures of the invention(or interior, fluid-contacting surfaces) from oxidized silicone polymersis that these surfaces can be much more hydrophilic than the surfaces oftypical elastomeric polymers (where a hydrophilic interior surface isdesired). Such hydrophilic channel surfaces can thus be more easilyfilled and wetted with aqueous solutions than can structures comprisedof typical, unoxidized elastomeric polymers or other hydrophobicmaterials.

In one embodiment, a bottom wall is formed of a material different fromone or more side walls or a top wall, or other components. For example,the interior surface of a bottom wall can comprise the surface of asilicon wafer or microchip, or other substrate. Other components can, asdescribed above, be sealed to such alternative substrates. Where it isdesired to seal a component comprising a silicone polymer (e.g. PDMS) toa substrate (bottom wall) of different material, the substrate may beselected from the group of materials to which oxidized silicone polymeris able to irreversibly seal (e.g., glass, silicon, silicon oxide,quartz, silicon nitride, polyethylene, polystyrene, epoxy polymers, andglassy carbon surfaces which have been oxidized). Alternatively, othersealing techniques can be used, as would be apparent to those ofordinary skill in the art, including, but not limited to, the use ofseparate adhesives, thermal bonding, solvent bonding, ultrasonicwelding, etc.

Various aspects and embodiments of the present invention are illustratedin the following examples. It will be appreciated that modification ofdetail may be made without departing from the scope of the invention.

EXAMPLES Example 1 Microfluidic Device for Combinatorial Chemistry andScreening Using In Vitro Compartmentalisation

A schematic representation of the microfluidic device is shown in FIG.15. Microchannels are fabricated with rectangular cross-sections usingrapid prototyping in poly(dimethylsiloxane) (PDMS) (McDonald andWhitesides, 2002) and rendered hydrophobic as (Song and Ismagilov,2003). Syringe pumps were used to drive flows (Harvard Apparatus PHD2000 Infusion pumps). For aqueous solutions, 250 μl Hamilton Gastightsyringes (1700 series, TLL) with removable needles of 27-gauge are usedwith 30-gauge Teflon tubing (Weico Wire and Cable). For the carrierfluid, 1 ml Hamilton Gastight syringes (1700 series, TLL) are used with30-gauge Teflon needles with one hub from Hamilton (Song and Ismagilov,2003). The carrier fluid is 9% (v/v) C₆F₁₁C₂H₄OH in perfluorodecaline(PFD) (Song et al., 2003). The microfluidic device consists of a seriesof interconnected modules. Each module has a specific function. Theseinclude modules that will produce droplets, fuse droplets, mix droplets,react droplets, detect droplets, and sort droplets (see FIG. 15). In oneexample, droplets are made, consisting of different molecules ordifferent concentrations of molecules.

Droplets are made at rates of up to 10⁴ sec⁻¹, and are made with apolydispersity of less than 1.5% and with sizes ranging from 1 μm to 100μm. Each droplet is fused with a second droplet containing a second setof reactants, and is rapidly mixed to initiate the chemical reaction.This chemical reaction is allowed to proceed in each droplet by passingit through a delay channel. Each droplet is then fused with anotherdroplet containing a second set of reactants, and is subsequentlyrapidly mixed to initiate the second set of chemical reactions. Afterthe second reaction has proceeded in a delay module, the results of thereaction is determined using an optical sensor or other form ofdetection module. Finally, the desired droplets are sorted into twopopulations based on signal from the optical detection module, onepopulation is kept for further processing and the other discarded. Theseand other modules can be used in this combination, or in othercombinations.

Droplet generation module: We use a flow-focusing geometry to form thedrops. A water stream is infused from one channel through a narrowconstriction; counter propagating oil streams hydrodynamically focus thewater stream reducing its size as it passes through the constriction asshown in FIG. 23A. This droplet generator can be operated in a flowregime that produces a steady stream of uniform droplets of water inoil. The size of the water droplets is controlled by the relative flowrates of the oil and the water; the viscous forces overcome surfacetension to create uniform droplets. If the flow rate of the water is toohigh a longer jet of fluid passes through the orifice and breaks up intodroplets further down stream; these droplets are less uniform in size.If the flow rate of the water is too low, the droplet breakup in theorifice becomes irregular again, producing a wider range of dropletsizes. While this emulsification technology is robust, it is limited toproducing droplets of one size at any given flow rate; this droplet sizeis largely determined by the channel dimensions. Moreover, the timing ofthe droplet production cannot be controlled.

We overcome these limitations by incorporating electric fields to createan electrically addressable emulsification system. To achieve this, weapply high voltage to the aqueous stream and charge the oil waterinterface, as shown schematically in FIG. 23A. The water stream behavesas a conductor while the oil is an insulator; electrochemical reactionscharge the fluid interface like a capacitor. At snap-off, charge on theinterface remains on the droplet. In addition, the droplet volume,V_(d), and frequency, f, can be tailored over nearly three orders ofmagnitude without changing the infusion rate of the oil or water.Droplet size and frequency are not independent; instead their product isdetermined by the infusion rate of the dispersed phase Q_(d)=f V_(d).The droplet size decreases with increasing field strength, as shown inFIGS. 23, B to E. The dependence of the droplet size on applied voltagefor three different flow rates is summarized in FIG. 23F. At low appliedvoltages the electric field has a negligible effect, and dropletformation is driven exclusively by the competition between surfacetension and viscous flow. By contrast, at high electric field strengths,there is a significant additional force on the growing drop, F=qE, whereq is the charge on the droplet. Since the droplet interface behaves as acapacitor, q is proportional to the applied voltage, V. This leads to aV² dependence of the force, which accounts for the decrease in dropletsize with increasing applied field shown in FIG. 23F. If the electricfield becomes too large, the charged interface of the water stream isrepelled by the highly charged drops; this destabilizes the productionand increases the variation in droplet size.

The electronic control afforded by the field-induced droplet formationprovides an additional valuable benefit: it allows the phase of thedroplet break-off to be adjusted within the production cycle. This isaccomplished by increasing the field above the critical break-off fieldonly at the instant the droplet is required. This provides a convenientmeans to precisely synchronize the production and arrival of individualdroplets at specific locations.

Droplet coalescer module: An essential component in any droplet-basedreaction-confinement system is a droplet coalescing module whichcombines two or more reagents to initiate a chemical reaction. This isparticularly difficult to achieve in a microfluidic device becausesurface tension, surfactant stabilization, and drainage forces allhinder droplet coalescence; moreover, the droplets must cross the streamlines that define their respective flows and must be perfectlysynchronized to arrive at a precise location for coalescence.

Use of electrostatic charge overcomes these difficulties; placingcharges of opposite sign on each droplet and applying an electric fieldforces them to coalesce. As an example we show a device consisting oftwo separate nozzles that generate droplets with different compositionsand opposite charges, sketched in FIG. 24A. The droplets are broughttogether at the confluence of the two streams. The electrodes used tocharge the droplets upon formation also provide the electric field toforce the droplets across the stream lines, leading to coalesce. Slightvariations in the structure of the two nozzles result in slightdifferences in the frequency and phase of their droplet generation inthe absence of a field. Thus the droplets differ in size even though theinfusion rates are identical. Moreover, the droplets do not arrive atthe point of confluence at exactly the same time. As a result thedroplets do not coalesce as shown in FIG. 24B. By contrast, uponapplication of an electric field, droplet formation becomes exactlysynchronized, ensuring that pairs of identically sized droplets eachreach the point of confluence simultaneously. Moreover, the droplets areoppositely charged, forcing them to traverse the stream lines andcontact each other, thereby causing them to coalesce, as shown in FIG.24C. The remarkable synchronization of the droplet formation resultsfrom coupling of the break-off of each of the pair of droplets asmediated by the electric field; the magnitude of the electric fieldvaries as the separation between the leading edges of the two dropletschanges and the frequency of droplet break-off is mode-locked to theelectric field. A minimum charge is required to cause droplets tocoalesce, presumably because of the stabilizing effects of thesurfactant coating; this is clear from FIG. 24D which shows the voltagedependence of the percentage of drops that contact each other thatactually coalesce.

Droplet mixer module: Rapid mixing is achieved through either successiveiterations of translation and rotation, FIG. 25, or by coalescing dropsalong the direction parallel to the flow direction, FIG. 26.

Droplet reactor/time delay module: A delay line is used to provide afixed time for a reaction. Two non-limiting examples of how this can beachieved are ‘single file’ and ‘large cross-section’ channels. The‘single file’ delay line uses length to achieve a fixed reaction time.As this often results in exceptionally long channels, it is desirable toplace spacer droplets of a third fluid, immicible with both the carrieroil and the aqueous droplets inbetween aqueous droplet pairs. There isthen an alternation between aqueous and non-aqueous droplets in acarrier oil. This is shown in FIG. 27A. A second possibility forachieving a long time delay is to use wide and deap channel having a‘large cross-sectional area’ to slow the average velocity of thedroplets. An example of this is shown in FIG. 27B.

Recharging module: The use of oppositely charged droplets and anelectric field to combine and mix reagents is extremely robust, and 100%of the droplets coalesce with their partner from the opposite stream.However, after they coalesce the resultant drops carry no electrostaticcharge. While it is convenient to charge droplets during formation,other methods must be employed in any robust droplet-based microfluidicsystem to recharge the mixed droplets if necessary for furtherprocessing. This is readily accomplished through the use of extensionalflow to split neutral droplets in the presence of an electric fieldwhich polarizes them, resulting in two oppositely charged daughterdroplets; this is sketched in FIG. 28A. The photomicrograph in FIG. 28Bshows neutral droplets entering a bifurcation and splitting into chargeddaughter droplets. The dashed region in FIG. 28B is enlarged in FIG. 28Cto illustrate the asymmetric stretching of the charged droplets in theelectric field. The vertical dashed lines indicate the edges of theelectrodes where the droplets return to their symmetric spherical shape.The electric field also allows precision control of the dropletsplitting providing the basis for a robust droplet division module whichallows the splitting of the contents into two or more aliquots ofidentical reagent, facilitating multiple assays on the contents of thesame microreactor.

Detection module: The detection module consists of an optical fiber, oneor more laser, one or more dichroic beam splitter, bandpass filters, andone or more photo multiplying tube (PMT) as sketched in FIG. 29.

Sorting module: The contents of individual droplets must be probed, andselected droplets sorted into discreet streams. The use of electrostaticcharging of droplets provides a means for sorting that can be preciselycontrolled, can be switched at high frequencies, and requires no movingparts. Electrostatic charge on the droplets enables drop-by-drop sortingbased on the linear coupling of charge to an external electric field. Asan example, a T-junction bifurcation that splits the flow of carrierfluid equally will also randomly split the droplet population equallyinto the two streams, as shown in FIG. 30A. However, a small electricfield applied at the bifurcation precisely dictates which channel thedrops enter; a schematic of the electrode configuration is shown in FIG.30B. Varying the direction of the field varies the direction of thesorted droplets as shown in FIGS. 30C and 30D. The large forces that canbe imparted on the droplets and the high switching frequency make this afast and robust sorting engine with no moving parts; thus the processingrate is limited only by the rate of droplet generation.

Example 2

Screening for Protein Tyrosine Phosphatase 1B (PTP1B) Inhibitors UsingMicrocapsules in Microfluidic Systems

PTP1B is a negative regulator of insulin and leptin signal transduction.Resistance to insulin and leptin are hallmarks of type 2 diabetesmellitus and obesity and hence PTP1B is an attractive drug target fordiabetes and obesity therapy (Johnson et al., 2002). Using amicrofluidic device as described in Example 1, we describe how PTP1Binhibitors can be screened using microcapsules in a microfluidic system.

All water-soluble reagents are dissolved in (25 mM HEPES, pH 7.4, 125 mMNaCl, 1 mM EDTA), a buffer compatible with PTP1B activity. A solution ofthe target enzyme (human recombinant PTP1B, residues 1-322; BiomolResearch Laboratories, Inc.) at 50 mU/ml and a solution of either a) 100μM compound 2 (FIG. 17), which has a bis-difluoromethylene phosphonateand is a known PTP1B inhibitor (Johnson et al., 2002), or b) 100 μMhydrocinnamic acid (Aldrich), a compound that is not a PTP1B inhibitorare compartmentalised into microcapsules using the device. Eachmicrocapsule containing target enzyme is fused with a microcapsulecontaining compound 2 or a microcapsule containing hydrocinnamic acid.Microcapsules containing either compound 2 or hydrocinnamic acid can beformed by switching between injection with syringes containing compound2 and hydrocinnamic acid.

After microcapsule fusion the contents are rapidly mixed. After thispoint the microcapsules are run for up to 1 min through a 60 cm longmicrochannel (to allow inhibitor binding). This microchannel is thenmerged with a second microchannel containing aqueous microcapsulescontaining the fluorogenic PTP1B substrate6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) (Molecular Probes)in 25 mM HEPES, pH 7.4, 125 mM NaCl, 1 mM EDTA and the microcapsulesfused pairwise. The fused microcapsules are then run for up to 2 minthrough a 60 cm long microchannel. Fluorescence of the microcapsules dueto production of DiFMU (excitation/emmission maxima 358/452 nm; bluefluorescence) is measured. Predominantly, microcapsules exhibiting bluefluorescence are those containing hydrocinnamic acid whereasmicrocapsules containing compound 2 exhibit low fluorescence due toinhibition of PTP1B.

Example 3 Screening of PTP1B Inhibitors from a Compound Library

96 aqueous mixtures are made on ice (to prevent reaction). The firstmixture contains 100 μM compound 2 (FIG. 17), which has abis-difluoromethylene phosphonate and is a known PTP1B inhibitor(Johnson et al., 2002), and a pre-defined ratio of Qdot.™. StreptavidinConjugates with emmission maxima at 585 nm, 655 nm and 705 nm (QuantumDot Corporation, Hayward Calif.) in a buffer compatible with PTP1Bactivity (25 mM HEPES, pH 7.4, 125 mM NaCl, 10% glycerol, 1 mM EDTA)(Doman et al., 2002). The 95 other aqueous mixtures are identical to theabove but each contain one of 95 carboxylic acids from the CarboxylicAcid Organic Building Block Library (Aldrich) in place of compound 2,and different ratios of Qdot.™. Streptavidin Conjugates with emissionmaxima at 585 nm, 655 nm and 705 nm. In all mixtures the concentrationof the 705 nm Qdot.™. Streptavidin Conjugates is 100 nM, and theconcentrations of the 585 nm and 655 nm Qdot.™. Streptavidin Conjugatesis either 0, 11, 22, 33, 44, 55, 66, 77, 88 or 100 nM. Hence, there are100 (10×10) permutations of Qdot.™. Streptavidin Conjugateconcentrations which allows the microcapsules containing each compoundto have a unique fluorescence signature which is read by determining thefluorescence ratio of fluorescence at 705 nm, 585 nm and 655 nm.

The 96 mixtures are distributed into 96 wells of a microtitre plate.Aliquots from each well of the plate are loaded sequentially into themicrofluidic device described in Example 1 using thin tubes connected tothe microfluidic device which are dipped into reservoirs containing thedesired compounds, and capillary action is used to draw the desiredcompound from the reservoir into the microfluidic device. The mixturesare compartmentalised into microcapsules in the device. Eachmicrocapsule is fused with another microcapsule containing the targetenzyme (human recombinant PTP1B, residues 1-322; Biomol ResearchLaboratories, Inc.) at 5 mU/ml and rapidly mixed. After incubating for10 min, at 37° C. in a delay line the microcapsule is fused with afurther microcapsule containing the fluorogenic PTP1B substrate6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) (Molecular Probes),and incubated at 37° C. for 30 min. in a delay line. Inhibitors reducethe amount of non-fluorescent substrate (DiFMUP) converted to thedephosphorylated product (DiFMU; excitation/emmission maxima 358/452 nm;blue fluorescence). Microcapsule fluorescence is then analysed.Predominantly, all microcapsules exhibited blue fluorescence due todephosphorylation of DiFMUP by PTP1B except those with the Qdotfluorescence signature of the microcapsules containing compound 2.

Example 4 Attachment of a Compound Library to Microbeads

5.5 μm diameter polystyrene microbeads that bear carboxylate functionalgroups on the surface are commercially available (www.luminexcorp.com)in an optically tagged form, as a result of incorporation of preciseratios of orange (585 nm), and red (>650 nm) fluorochromes (Fulton etal., 1997). A set of 100 such beads, each with a unique opticalsignature (www.luminexcorp.com) are modified with an excess ofethylenediamine and EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride (Pierce) as (Hermanson, 1996) to create primary aminogroups on the surface. The photocleavable linker4-(4-hydroxymethyl-2-methoxy-5-nitrophenoxy)butanoic acid (NovaBiochem)(Holmes and Jones, 1995) is then attached to the beads by forming anamide bond using EDC as above. 100 different carboxylic acids from theCarboxylic Acid Organic Building Block Library (Aldrich) are thencoupled to the beads, by reacting with the linker alcohol to form acarboxylate ester, each of the 100 different optically tagged beadsbeing coupled to a different carboxylic acid, and each bead beingderivatised with ˜10⁶ molecules of carboxylic acid. Irradiation for 4min on ice using a B100 AP 354 nm UV lamp (UVP) from a distance of ˜5 cmresults in release of the compounds from the beads as carboxylic acids.

Example 5 Screening for Inhibitors of the Enzyme Protein TyrosinePhosphatase 1B (PTP1B) Using Compounds Attached to Microbeads

5.5 μm diameter polystyrene microbeads that bear carboxylate functionalgroups on the surface are commercially available (www.luminexcorp.com)in an optically tagged form, as a result of incorporation of preciseratios of orange (585 nm), and red (>650 nm) fluorochromes (Fulton etal., 1997). First, the carboxylate functional groups on the microbeadsare converted to primary amines using ethylenediamine and EDC as inexample 4. A phosphopeptide substrate for PTP1B, the undecapaptideEGFR₉₈₈₋₉₉₈ (DADEPYLIPQQG) (Zhang et al., 1993), is then coupled to bothsets of microbeads via the surface amino groups using EDC. This peptideis made by solid phase synthesis on Sieber Amide resin(9-Fmoc-amino-xanthen-3-yloxy-Merrifield resin) (Novabiochem) withorthogonal protection on the side chain carboxylate groups usingcarboxylate-O-allyl esters. A linker comprised of tetradecanedioic acidis coupled to the N-terminus and the peptide cleaved from the beadsusing 1% TFA to yield a peptide with a C-terminal amide. The peptide iscoupled to the beads (using EDC) via the linker to give ˜10⁵ peptidesper bead. The remaining surface amino groups are then modified byattaching the photochemically cleavable linker4-(4-hydroxymethyl-2-methoxy-5-nitrophenoxy)butanoic acid as in example4. The protecting groups on the side chain carboxylates of the peptideare then removed using Pd(Ph₃)₄/CHCl₃/HOAc/N-methyl morpholine. A firstset of microbeads is derivatised with3-(4-difluorophosphonomethylphenyl)propanoic acid (compound 1, FIG. 17),a compound that is a known PTP1B inhibitor (Johnson et al., 2002). Asecond set of beads, with a distinct optical tag from the first set ofbeads, is derivatised with hydrocinnamic acid (Aldrich), a compound thatis not a PTP1B inhibitor. In each case the compound is coupled byreacting with the linker alcohol to form a carboxylate ester as inexample 4. Each microbead is derivatised with ˜10⁶ molecules (Fulton etal., 1997).

The microbeads are then screened using the microfluidic system outlinedin FIG. 15. The two sets of microbeads are mixed in ratios varying from1:1000 to 1:1 (compound 1 beads: hydrocinnamic acid beads) and 10⁸ totalmicrobeads are mixed with the target enzyme (human recombinant PTP1B,residues 1-322; Biomol Research Laboratories, Inc.) at a concentrationof 10 nM, on ice (to prevent reaction) in a buffer compatible with PTP1Bactivity (25 mM HEPES, pH 7.4, 125 mM NaCl, 10% glycerol, 1 mM EDTA)(Doman et al., 2002). Single beads and target enzyme (PTP1B) are thencolocalised in microcapsules by forming microcapsules using themicrofluidic system described in Example 1. The concentration of beadsis such that most microcapsules contain one or no beads. Eachmicrocapsule is fused with another microcapsule containing the targetenzyme (human recombinant PTP1B, residues 1-322; Biomol ResearchLaboratories, Inc.) at 5 mU/ml and rapidly mixed. The compound isreleased photochemically (as in example 4). After incubating for 10 min.at 37° C. in a delay line the microcapsule is fused with a furthermicrocapsule containing the fluorogenic PTP1B substrate6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) (Molecular Probes),and incubated at 37° C. for 30 min. in a delay line. Inhibitors reducethe amount of substrate converted to product (dephosphorylated peptide).The microcapsules are collected, cooled to 4° C. and broken as(Griffiths and Tawfik, 2003) into 100 μM vanadate to stop the reaction(Harder et al., 1994). After labelling with an anti-substrate(anti-phosphotyrosine) antibody labelled with the green (530 nm)fluorochrome fluorescein isothiocyanate (mouse monoclonal IgG_(2b) PY20(Santa Cruz) according to the manufacturer's instructions, beads areanalysed by 3-colour flow cytometry using a FACScan (Becton-Dickinson),FACScalibur (Becton-Dickinson) or MoFlo (Cytomation) flow cytometers tosimultaneously determine the extent of inhibition and the compound onthe beads. Predominantly, dephosphorylation of the peptide is onlyobserved on those microbeads which were coated with PTP1B inhibitors,and not on other microbeads.

Example 6 Screening of PTP1B Inhibitors from a Compound Library Attachedto Microbeads

A set of 100 5.5 μm diameter polystyrene microbeads, bearing carboxylatefunctional groups on the surface and each with a unique opticalsignature (www.luminexcorp.com) as a result of incorporation of preciseratios of orange (585 nm), and red (>650 nm) fluorochromes (Fulton etal., 1997) are derivatised with a phosphopeptide substrate for PTP1B,the undecapaptide EGFR₉₈₈₋₉₉₈ (DADEpYLIPQQG) (Zhang et al., 1993), and100 different carboxylic acids, each attached via a photochemicallycleavable linker, as in example 4. One of these carboxylic acids is3-(4-difluorophosphonomethylphenyl) propanoic acid (compound 1, FIG.17), a compound that is a known PTP1B inhibitor (Johnson et al., 2002).The other 99 carboxylic acids are from the Carboxylic Acid OrganicBuilding Block Library (Aldrich) as example 4. Equal numbers of each ofthe 100 bead sets are then mixed and screened as for example 5.Predominantly, dephosphorylation of the peptide is only observed onthose microbeads which were coated with the PTP1B inhibitor3-(4-difluorophosphonomethylphenyl) propanoic acid (compound 1, FIG.17), and not on microbeads coated with other compounds.

Example 7 Compartmentalisation of Small Molecules in aWater-in-Fluorocarbon Emulsions

Water-in-fluorocarbon emulsions containing 95% (v/v) perfluorooctylbromide, 5% (v/v) phosphate buffered saline containing the molecule ofinterest in solution, and 2% (w/v) C₈F₁₇C₁₁H₂₂OP(O)[N(CH₂CH₂)₂O]₂(F8H11DMP) as surfactant were formed essentially as (Sadtler et al.,1996) by extrusion (15 times) through 14 μm filters (Osmonics) or byhomogenising for 5 min at 25,000 r.p.m. using an Ultra-Turrax T8Homogenizer (IKA) with a 5 mm dispersing tool. Emulsions were madecontaining a series of small fluorescent molecules dissolved in theaqueous phase at concentrations from 100 μm to 2 mM. These molecules,including calcein, texas red, fluorescein, coumarin 102,7-hydroxycoumarin-3-carboxylic acid and 7-diethylamino-4-methyl coumarin(coumarin 1), had molecular weights from 203 to 625 Da and LogPvalues—calculated using SRC's LogKow/KowWin Program (Meylan and Howard,1995)—ranging from −0.49 to 4.09. Emulsions containing differentcoloured fluorochromes were mixed by vortexing. Compartmentalisation wasobserved by epifluorescence microscopy of the mixed emulsions. Noexchange between compartments was observed 24 hours after mixing (seeFIG. 19).

Example 8 Compartmentalisation of Small Molecules in aWater-in-Fluorocarbon Emulsions Made Using Microfluidic Systems

Water-in-fluorocarbon emulsions containing 95% (v/v) perfluorooctylbromide, 5% (v/v) phosphate buffered saline containing the molecule ofinterest in solution, and 2% (w/v) C₈F₁₇C₁₁H₂₂OP(O)[N(CH₂CH₂)₂O]₂(F8H11DMP) as surfactant were formed essentially using multiple dropletgeneration modules as described in Example 1. The aqueous phase at eachof the nozzles contained a different small fluorescent moleculesdissolved at concentrations from 100 μM to 2 mM. These molecules,including calcein, texas red, fluorescein, coumarin 102,7-hydroxycoumarin-3-carboxylic acid and 7-diethylamino-4-methyl coumarin(coumarin 1), had molecular weights from 203 to 625 Da and LogPvalues—calculated using SRC's LogKow/KowWin Program (Meylan and Howard,1995)—ranging from—0.49 to 4.09. Emulsions containing different colouredfluorochromes were mixed by combining the streams carrying the droplethaving different fluorofers into a single stream containing all types ofdroplets. The stream carrying the collection of droplets then emptiesinto a deep well on the device where the droplets can be stored in closeproximity and monitored over time up to 24 hours. No cross contaminationbetween the droplets is observed.

Example 9 Determining Mode of Inhibition and K_(i) of PETG onβ-Galactosidase

Using a microfluidic device as described in Example 1, we demonstratethat the mode of inhibition of the enzyme E. coli β-galactosidase(LacZ), by phenylethyl β-D-thiogalactopyranoside (PETG), is competitive,and we show how we can obtain the inhibition constant (K_(i)) of PETG.In the enzyme inhibition assay, the rate of catalysis is determined byusing a non-fluorescent substrate for LacZ, fluoresceinmono-β-D-galactoside (FMG), and measuring the appearance of thefluorescent product, fluorescein (excitation 488 nm, emission 514 nm).All components of the LacZ inhibition assay are dissolved in assaybuffer (10 mM MgCl₂, 50 mM NaCl, 1 mM DTT, 100 μg/ml BSA, 10 mMTris-HCl, pH 7.9.

Leading into each droplet forming module (FIG. 15) are two Teflon tubesleading from syringe pumps. The channels leading from each tube merge tocreate a single flow before entering the droplet forming module. The twosyringes feeding into the first droplet forming module contain, (a) 50μM PETG in assay buffer, and (b), assay buffer. The two syringes feedinginto the second droplet forming module contain, (c) 100 nM LacZ in assaybuffer and (d), assay buffer. The two syringes feeding into the thirddroplet forming module contain, (e) 5 mM FMG in assay buffer, and (f),assay buffer. The final concentration of each component in each dropletis independently controllable by adjusting relative flow rates of eachcomponent and buffer solution while maintaining a constant combined flowrate from both syringes.

The first droplet fusion mixes the inhibitor (PETG) with the enzyme(LacZ). After the combined droplet has spent two minutes in a delay lineit is fused with a third droplet containing the fluorogenic enzymesubstrate (FMG). Finally, after all the components are mixed thereaction is observed by measuring fluorescence of individual droplets orby integrating fluorescent light from droplets with the sameconcentration of each component during 10-second exposure time atmultiple points in the second 10 min long delay line. Each fluorescenceintensity value at different positions is proportional to the amount ofproduct at different reaction times. Rate of product formation is linearduring initial reaction time and initial rate (v) can be determined fromlinear fitting. Data from the repeated measurement at differentconcentration of FMG and PETG are expressed in a Lineweaver-Burk plot(1/v vs. 1/[S]; where [S]=substrate concentration). The same y-interceptvalues at different concentration of PETG show that the mode of PETGinhibition is competitive. In competitive inhibition, each slope dividedby the y-intercept represents an apparent Michaelis-Menten constant thatis a linear function of the concentration of inhibitor. The y-interceptin a graph of apparent Michaelis-Menten constant versus competitiveinhibitor concentration gives the Michaelis-Menten constant, and theinverse of its slope multiplied by the y-intercept is K_(i). Using thefollowing conditions (in the final fused microcapsule), 30 nM LacZ, 0 to13 μM PETG and 10 to 700 μM FMG the K_(M) of FMG for LacZ can bedetermined to within 20% of the previously published value (118 μM;Huang, 1991), and the K_(i) of PETG for LacZ can be determined to bewithin the range of the previously published value (0.98 μM; Huang,1991).

Example 10

Synthesis of Secondary Compounds in Emulsion Microcapsules and Screeningfor PTP1B Inhibition in Microfluidic Systems

Using a microfluidic device as described in Example 1, we describe howPTP1B inhibitors can be synthesised and screened using microcapsules ina microfluidic system.

All water-soluble reagents are dissolved in (25 mM HEPES, pH 7.4, 125 mMNaCl, 1 mM EDTA), a buffer compatible with PTP1B activity. A compoundwhich is a primary amine is compartmentalised into microcapsules usingthe device. A second solution, of a compound which is an aldehyde isalso compartmentalised into microcapsules using the device. The aminesand aldehydes can either a) contain a difluoromethylene phosphonatemoiety (FIG. 22, compounds A and B), or b) contain no difluoromethylenephosphonate moiety. Each microcapsule containing a primary amine isfused with a microcapsule containing an aldehyde. Microcapsulescontaining compounds with and without difluoromethylene phosphonatemoieties can be formed by switching between injection with syringescontaining amines or aldehydes with or without a difluoromethylenephosphonate moiety.

After microcapsule fusion the contents are rapidly mixed and themicrocapsules pass down a delay line. This allows the amine and thealdehyde to react together by formation of a Schiff base to create asecondary compound and allows inhibitors to bind to PTP1B. Eachmicrocapsules are then fused with a further microcapsule containing asolution of the target enzyme (human recombinant PTP1B, residues 1-322;Biomol Research Laboratories, Inc.) at 50 mU/ml, and the fluorogenicPTP1B substrate 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP)(Molecular Probes). The fused microcapsules then run along a furtherdelay line. Fluorescence of the microcapsules due to production of DiFMU(excitation/emmission maxima 358/452 nm; blue fluorescence) is measured.

Predominantly, when the amine and aldehyde concentrations are low (<100μM) inhibition of PTP1B activity is only observed, in microcapsulescontaining both an amine with a difluoromethylene phosphonate moiety(compound A,) and an aldehyde with a difluoromethylene phosphonatemoiety (compound B,). This is because the Schiff base formed in thesemicrocapsules (compound C,) contains bis-difluoromethylene phosphonateand is a much more potent PTP1B inhibitor than a molecule with a singledifluoromethylene phosphonate moiety.

Predominantly, when the amine and aldehyde concentrations are high (>100μM) inhibition of PTP1B activity is observed in microcapsules containingeither an amine with a difluoromethylene phosphonate moiety (compound A,or an aldehyde with a difluoromethylene phosphonate moiety (compoundB,), or both, but not in other microcapsules. This is because at higherconcentrations molecules with either a single difluoromethylenephosphonate moiety or a bis-difluoromethylene phosphonate (compound C,FIG. 22) can inhibit PTP1B.

Example 11 Synthesis of a Library of 2304 Secondary Compounds inEmulsion Microcapsules and Screening of PTP1B Inhibition in MicrofluidicSystems

96 aqueous mixtures are made in a buffer compatible with PTP1B activity(25 mM HEPES, pH 7.4, 125 mM NaCl, 10% glycerol, 1 mM EDTA) (Doman etal., 2002) and containing the target enzyme (human recombinant PTP1B,residues 1-322; Biomol Research Laboratories, Inc.) at a concentrationof 5 mU/ml. The first 48 mixtures each contain a unique compoundcontaining a primary amine. One of these compounds (compound A, FIG. 22)contains a difluoromethylene phosphonate moiety. The second 48 mixturesof microbeads each contain a unique compound containing an aldehyde. Oneof these compounds (compound B, FIG. 22) contains a difluoromethylenephosphonate moiety.

The 96 mixtures are distributed into 96 wells of a microtitre plate.Aliquots from each well of the plate are loaded sequentially into themicrofluidic device described in Example 1 using thin tubes connected tothe microfluidic device which are dipped into reservoirs containing thedesired compounds, and capillary action is used to draw the desiredcompound from the reservoir into the microfluidic device. The mixturesare compartmentalised into microcapsules in the device.

Each microcapsule containing an amine is fused with a microdropletcontaining an aldehyde and incubated for 10 min, at 37° C. in a delayline. The amine and the aldehyde react together by forming a Schiffbase, resulting in the creation of a new molecule (an imine) insolution. The microcapsule is then fused with a further microcapsulecontaining the fluorogenic PTP1B substrate6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) (Molecular Probes),and incubated at 37° C. for 30 min. in a delay line. Inhibitors reducethe amount osf non-fluorescent substrate (DiFMUP) converted to thedephosphorylated product (DiFMU; excitation/emmission maxima 358/452 nm;blue fluorescence). Microcapsule fluorescence is then analysed.

Microcapsules containing a primary compound which is itself a PTP1Binhibitor, or which reacts with another primary compound to form asecond inhibitor are identified as little substrate has been convertedto product. When the compounds are present in the microcapsules at highconcentration, the identified microcapsules where little substrate hasbeen converted to product include those carrying compounds containing adifluoromethylene phosphonate moiety. When the compounds are present inthe microcapsules at low concentration, the concentration of thecompounds containing a difluoromethylene phosphonate moieties in themicrocapsules is insufficient to efficiently inhibit PTP1B (see example10). However, in microcapsules where two primary compounds have reactedto form an imine containing a bis-difluoromethylene phosphonate moiety,which is a highly potent PTP1B inhibitor (compound C, FIG. 22), thisinhibits conversion of the PTP1B substrate to product and littlefluorescence is observed.

Example 12

Synthesis of Secondary Compounds in Emulsion Microcapsules and Screeningfor PTP1B Inhibition Using Compounds Attached to Microbeads inMicrofluidic Systems

5.5 μm diameter polystyrene microbeads that bear carboxylate functionalgroups on the surface are commercially available (www.luminexcorp.com)in an optically tagged form, as a result of incorporation of preciseratios of orange (585 nm), and red (>650 nm) fluorochromes (Fulton etal., 1997). First, the carboxylate functional groups on the microbeadsare converted to primary amines using ethylenediamine and EDC as inexample 4. A phosphopeptide substrate for PTP1B, the undecapaptideEGFR₉₈₈₋₉₉₈ (DADEpYLIPQQG) (Zhang et al., 1993), is then coupled to bothsets of microbeads via the surface amino groups using EDC and theprotecting groups on the side chain carboxylates of the peptide removedas in example 5. A first set of microbeads (set 1) is reacted withsuccinimidyl p-formylbenzoate to convert the surface amino groups toaldehydes. A second set of microbeads (set 2), with a distinct opticaltag from the first set of microbeads, is left unreacted (i.e. withprimary amines on the surface).

The first set of microbeads (set 1), are then reacted with a compoundcontaining a difluoromethylene phosphonate moiety and a primary amine(compound A, FIG. 22) via reaction with the surface aldehyde groups toform a Schiff base. The second set of microbeads (set 2), with adistinct optical tag from the first set of beads, are reacted with acompound containing a difluoromethylene phosphonate moiety and analdehyde (compound B, FIG. 22) via reaction of the aldehyde with thesurface amine groups to form a Schiff base. The formation of Schiffbases is enhanced by reaction at alkaline pH (i.e. pH9-10). Microbeadscoated with compounds at various densities are created.

The two sets of microbeads are seperately suspended in a buffercompatible with PTP1B activity (25 mM HEPES, pH 7.4, 125 mM NaCl, 10%glycerol, 1 mM EDTA) (Doman et al., 2002). Each set of microbeads iscompartmentalised into microcapsules in the device described inExample 1. The number of microbeads is varied such that, at one extreme,most microcapsules contain one or no beads, and at the other, themajority of microcapsules contain two or more microbeads. Eachmicrocapsule containing a set 1 microbead is fused with a microcapsulecontaining a set 2 microbead and incubated for 10 min, at 37° C. in adelay line. The Schiff base is a relatively labile, reversibleinteraction, readily hydrolysed at neutral pH, resulting in release ofcompounds from the beads. In microcapsules containing a microbead fromboth set 1 and set 2, the compounds released from the microbeads canreact with each other, forming a Schiff base and creating a new moleculein solution. This new molecule (FIG. 22, compound C) contains abis-difluoromethylene phosphonate moiety and has significantly morepotency as a PTP1B inhibitor than compounds with a singledifluoromethylene phosphonate moiety (see FIG. 22). The microcapsule isthen fused with a further microcapsule containing the target enzyme(human recombinant PTP1B, residues 1-322; Biomol Research Laboratories,Inc.) at a concentration of 5 mU/ml in 25 mM HEPES, pH 7.4, 125 mM NaCl,10% glycerol, 1 mM EDTA, and incubated at 37° C. for 30 min. in a delayline. Inhibitors reduce the amount of substrate converted to product(dephosphorylated peptide). The microcapsules are collected, cooled to4° C., and broken into 100 μM vanadate to stop the reaction (Harder etal., 1994). After labelling with an anti-substrate(anti-phosphotyrosine) antibody labelled with the green (530 nm)fluorochrome fluorescein isothiocyanate (mouse monoclonal IgG2b PY20(Santa Cruz) according to the manufacturer's instructions, beads areanalysed by 3-colour flow cytometry using a FACScan (Becton-Dickinson),FACScalibur (Becton-Dickinson) or MoFlo (Cytomation) flow cytometers tosimultaneously determine the extent of inhibition and the compound onthe beads. With low microbead numbers, most microcapsules contain only asingle or no microbeads and PTP1B inhibition is only detected on beadscoated with a high density of inhibitor, when the concentration ofinhibitor released into solution in each microcapsule is sufficientlyhigh for effective inhibition. In contrast, when the bead numbers arehigher, many microbeads are detected where little substrate has beenconverted to product, even when the microbeads are coated with inhibitorat low density. This is due to the formation of a highly potent PTP1Binhibitor (FIG. 22, compound C) containing a bis-difluoromethylenephosphonate moiety in microcapsules containing a microbead each from set1 and set 2.

Example 13

Synthesis of a Library of 2500 Secondary Compounds in EmulsionMicrocapsules and Screening for PTP1B Inhibition Using CompoundsAttached to Microbeads in Microfluidic Systems

A set of 100 5.5 μm diameter polystyrene micro beads, bearingcarboxylate functional groups on the surface and each with a uniqueoptical signature (www.luminexcorp.com) as a result of incorporation ofprecise ratios of orange (585 nm), and red (>650 nm) fluorochromes(Fulton et al., 1997) are modified to convert the carboxylate functionalgroups to primary amines as in example 4, then derivatised with aphosphopeptide substrate for PTP1B, the undecapaptide EGFR₉₈₈₋₉₉₈(DADEpYLIPQQG) (Zhang et al., 1993), as in example 5. The first 50 setsof microbeads are reacted to convert a proportion of the surfacecarboxyl groups to aldehydes as in example 10. The second 50 sets ofmicrobeads are left unreacted (i.e. with primary amines on the surface).

The first 50 sets of microbeads are each reacted with a unique compoundcontaining a primary amine via reaction with the surface aldehyde groupsto form a Schiff base which links the compounds to the beads. One ofthese compounds (compound A, FIG. 22) contains a difluoromethylenephosphonate moiety. The second 50 sets of microbeads are each reactedwith a unique compound containing an aldehyde via reaction with thesurface amine groups to form a Schiff base which links the compounds tothe beads. One of these compounds (compound B, FIG. 22) contains adifluoromethylene phosphonate moiety. The formation of Schiff bases isenhanced by reaction at alkaline pH (i.e. pH9-10).

The 50 sets of microbeads which were reacted with primary amines (aminemicrobeads) are pooled and suspended in a buffer compatible with PTP1Bactivity (25 mM HEPES, pH 7.4, 125 mM NaCl, 10% glycerol, 1 mM EDTA)(Doman et al., 2002). The 50 sets of microbeads which were reacted withaldehydes (aldehyde microbeads) are pooled and suspended in the samebuffer. The amine microbeads and the aldehyde microbeads arecompartmentalised into microcapsules in the device described inExample 1. The number of microbeads is set such that the majority ofmicrodroplets contain a single microbead. Each microcapsule containingan amine microbead is fused with a microcapsule containing an aldehydemicrobead and incubated for 10 min, at 37° C. in a delay line. Themicrocapsule is then fused with a further microcapsule containing thetarget enzyme (human recombinant PTP1B, residues 1-322; Biomol ResearchLaboratories, Inc.) at a concentration of 5 mU/ml in 25 mM HEPES, pH7.4, 125 mM NaCl, 10% glycerol, 1 mM EDTA, and incubated at 37° C. for30 min. in a delay line. The Schiff base is a relatively labile,reversible interaction, readily hydrolysed at neutral pH, resulting inrelease of compounds from the beads. In microcapsules containing amicrobead from one of the first 50 sets and a microbead from one of thesecond 50 sets, the compounds released from the microbeads can reactwith each other, forming a Schiff base and creating a new molecule insolution. Inhibitors reduce the amount of substrate converted to product(dephosphorylated peptide). The microcapsules are collected, cooled to4° C., and broken into 100 μM vanadate to stop the reaction (Harder etal., 1994). After labelling with an anti-substrate(anti-phosphotyrosine) antibody labelled with the green (530 nm)fluorochrome fluorescein isothiocyanate (mouse monoclonal IgG_(2b) PY20(Santa Cruz) according to the manufacturer's instructions, beads areanalysed by 3-colour flow cytometry using a FACScan (Becton-Dickinson),FACScalibur (Becton-Dickinson) or MoFlo (Cytomation) flow cytometers tosimultaneously determine the extent of inhibition and the compound onthe beads.

Beads which were coated with a primary compound which is itself a PTP1Binhibitor, or which reacts with another primary compound released fromanother co-compartmentalised bead to form a second inhibitor areidentified as little substrate has been converted to product. Theidentified beads where as little substrate has been converted to productinclude those carrying compounds containing a difluoromethylenephosphonate moiety. When the microbeads are coated with compounds at lowdensity the concentration of the released compounds containing adifluoromethylene phosphonate moieties in the microcapsules isinsufficient to efficiently inhibit PTP1B (see example 12). However inmicrocapsules containing two microbeads, one from the first set of 50beads and one from the second set of 50 beads, and where each microbeadcarries a molecule with a difluoromethylene phosphonate moiety, thereleased molecules can form a highly potent PTP1B inhibitor (compound C,FIG. 22) containing a bis-difluoromethylene phosphonate moiety in themicrocapsules which inhibits conversion of the PTP1B substrate toproduct.

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All publications mentioned in the above specification, and referencescited in said publications, are herein incorporated by reference.Various modifications and variations of the described methods and systemof the invention will be apparent to those skilled in the art withoutdeparting from the scope and spirit of the invention. Although theinvention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the followingclaims.

1. A method for preparing a repertoire of compounds comprising the stepsof: (a) compartmentalising two or more sets of primary compounds intomicrocapsules; such that a proportion of the microcapsules containsmultiple copies of one or more compounds representative of each of saidsets, and wherein said one or more compounds form a subset of the set ofprimary compounds; (b) forming secondary compounds in the microcapsulesby chemical reactions between primary compounds from different sets;wherein one or more of steps (a) and (b) are performed undermicrofluidic control.
 2. A method for identifying primary compoundswhich react together to form secondary compounds capable of binding toor modulating the activity of a target, comprising the steps of: (a)compartmentalising two or more sets of primary compounds intomicrocapsules; such that a proportion of the microcapsules contains twoor more compounds; (b) forming secondary compounds in the microcapsulesby chemical reactions between primary compounds from different sets; and(c) identifying subsets of primary compounds which react to formsecondary compounds which bind to or modulate the activity of thetarget; wherein one or more of steps (a) and (b) and (c) are performedunder microfluidic control.
 3. A method for synthesising compounds withenhanced ability to bind to or modulate the activity of the target,comprising the steps of: (a) compartmentalising into microcapsulessubsets of primary compounds identified in step (c) of the second aspectof the invention and, optionally, compartmentalising additional sets ofprimary compounds; (b) forming secondary compounds in the microcapsulesby chemical reactions between primary compounds from different sets; and(c) identifying subsets of primary compounds which react to formsecondary compounds which bind to or modulate the activity of thetarget; wherein one or more of steps (a) and (b) and (c) are performedunder microfluidic control.
 4. A method for identifying individualcompounds which bind to or modulate the activity of the target,comprising the steps of: compartmentalising into microcapsules a primarycompound identified in step (c) of the second or third aspect of theinvention and additional sets of primary compounds; forming secondarycompounds in the microcapsules by chemical reactions between primarycompounds from different sets; and identifying subsets of primarycompounds which react to form secondary compounds which bind to ormodulate the activity of the target; wherein one or more of steps (a)and (b) and (c) are performed under microfluidic control.